Medicinal composition of extract of seed of emblica officinalis and method of preparing the same

ABSTRACT

An extract of seeds of Emblica officinalis which has triterpenoids and hydroxycinnamic acids is provided. A blend of extracts of seeds of Emblica officinalis which includes an extract of seeds of Emblica officinalis having triterpenoids and hydroxycinnamic acids and an extract of seeds of Emblica officinalis having fatty acids, such as, alpha linolenic acid, linoleic acid and oleic acid is provided. Methods of preparing the extracts of seeds of Emblica officinalis and methods of preparing the blend of extracts of seeds of Emblica officinalis are provided. Methods of treatment administering the extracts of seeds of Emblica officinalis and methods of preparing the blend of extracts of seeds of Emblica officinalis are provided.

FIELD

The disclosure relates a medicinal composition of the extract of seed ofEmblica officinalis, a method of preparing a composition consisting ofextract of seed of Emblica officinalis more particularly which hasapplication as a nutraceutical or pharmaceutical for reducing the totalcholesterol, reducing triglyceride, reducing blood glucose level,enhancing HDL-Cholesterol, increasing the HDL-Cholesterol to totalcholesterol ratio, lowering LDL-Cholesterol levels, reducing the CRPlevel, decreasing the intima media thickening, reducing hair fall,reducing atherogenic index of plasma, increasing Apo A-I levels,decreasing Apo B levels, decreasing Apo B to Apo A-I ratio, reducingHomocysteine level, reduction in glycosylated Hb, modulating Thyroidstimulating Hormone (TSH) level, decrease in HMGCoA reductase activity,lowering total cholesterol without a change in CoQ10 levels in mammalsespecially human beings. The composition is effective even at lowerdosage for reducing the total cholesterol, reducing triglyceride,reducing blood glucose level, enhancing HDL-Cholesterol levels,increasing the HDL-Cholesterol to total cholesterol ratio, loweringLDL-Cholesterol levels, reducing the CRP level, decreasing the intimamedia thickening, reducing hair fall, reducing atherogenic index ofplasma, increasing Apo A-I levels, decreasing Apo B levels, decreasingApo B to Apo A-I ratio, reducing Homocysteine level, reduction inglycosylated Hb, modulating TSH level, decrease in HMGCoA reductaseactivity, lowering total cholesterol without a change in CoQ10 levels.

BACKGROUND

Amla (or Amlaka, Amlaki, or other variants) is one of the mostfrequently used of the Ayurvedic herbs; it is the fruit of Phyllanthusemblica, also called Emblica officinalis. The fruit is similar inappearance to the common gooseberry (Ribes spp., a type of currant),which is botanically unrelated to amla. However, due to the similarappearance of the fruit clusters, amla is usually called the “Indiangooseberry.” The plant, a member of the Euphorbiaceae, grows to become amedium-sized tree that is found growing in the plains and sub-mountainregions all over the Indian subcontinent from 200 to nearly 2000 metersabove sea level. Indian gooseberry is a wonder herbs and one of theprecious gifts of nature to man. It contributes towards health andlongevity.

Emblica officinalis (EO) enjoys a hallowed position in Ayurveda-anIndian indigenous system of medicine. According to ancient Indianmythology, it is the first tree to be created in the universe. Emblicaofficinalis fruit is one of the key constituents of the celebratedAyurvedic preparation, Chyavanaprash, used in India for thousands ofyears as a vitalizing and rejuvenating health tonic. According toAyurveda, amla balances all three doshas. While amla is unusual in thatit contains five out of the six tastes recognized by Ayurved, it is mostimportant to recognize the effects of the “virya”, or potency, and“vipaka”, or post-digestive effect. The fruits of EO are widely used inthe Ayurveda and are believed to increase defense against diseases. I

Coronary heart disease (CHD) continues to be the major cause ofpremature death in most developed and developing countries. A low levelof HDL cholesterol is the second most important risk factor for CHD, asdemonstrated in numerous clinical and epidemiological studies (Gorden,D. and Rifkind, H. M., N. Engl. J. Med., 1989, 321:1311-1315; Brewer,Jr., H. B., New Engl. J. Med, 2004, 350:1491-94) and HDL has emerged,during the past decade, as a new potential target for the treatment ofcardiovascular diseases. The key role of HDL as a carrier of excesscellular cholesterol in the reverse cholesterol transport pathway isbelieved to provide protection against atherosclerosis. In reversecholesterol transport, peripheral tissues, for example, vessel-wallmacrophages, remove their excess cholesterol through the ATP-bindingcassette transporter 1 (ABCA1) to poorly lipidated apolipoprotein A-I,forming pre-.beta.-HDL. Lecithin-cholesterol acyltransferase thenesterifies free cholesterol to cholesteryl esters, converting pre-β-HDLto mature spherical α-HDL.

HDL cholesterol is transported to the liver by two pathways: 1) it isdelivered directly to the liver through interaction with the scavengerreceptor, class B, type I (SR-BI); 2) cholesteryl esters in HDL aretransferred by the cholesterol ester transferase protein (CETP) tovery-low-density-lipoproteins (VLDL) and low-density lipoproteins (LDL)and are then returned to the liver through the LDL receptor. HDLcholesterol that is taken up by the liver is then excreted in the formof bile acids and cholesterol, completing the process of reversecholesterol transport (Brewer, H. B. Jr., Arterioscl. Thromb. Vasc.Biol., 2004, 24:387-91). HDL is believed to have the ability to removecholesterol from macrophages, thus preventing the formation of foamcells.

A second beneficial role of HDL in atherosclerosis is in protecting LDLfrom oxidation (Navab, M. et al, Circulation, 2002, 105:290-92). Unlikenormal LDL, oxidized LDL is readily taken up by macrophage scavengerreceptor SR-A or CD36 resulting in the formation of foam cells. Foamcells are a major component of the early atherosclerotic lesion.Further, HDL may slow the progression of lesions by selectivelydecreasing the production of endothelial cell-adhesion molecules thatfacilitate the uptake of cells into the vessel wall (Barter, P. J., etal, Curr. Opin. Lipid, 2002, 13:285-88). HDL may also prolong thehalf-life of prostacycline and preserve its vasodilatory effect(Mackness, M. I. et al, Atherosclerosis, 1993, 104:129-35).

Several lines of evidence support the concept that increasing the HDLlevel may provide protection against the development of atherosclerosis.Epidemiologic studies have shown an inverse relation between HDLcholesterol levels and the risk of cardiovascular disease. Increasingthe HDL cholesterol level by 1 mg may reduce the risk of cardiovasculardisease by 2 to 3 percent. Over expressing the apo-A-I gene intransgenic mice and rabbits and infusing complexes consisting of Apo A-1and phospholipids into hyperlipidemic rabbits increase HDL cholesterollevels and decrease the development of atherosclerosis (Brewer, H B,Jr., loc. cit). In humans, infusing either of these complexes orpro-apo-A-I results in short term increase in HDL cholesterol, biliarycholesterol and fecal cholesterol loss, reinforcing the concept thatelevating the HDL cholesterol level decreases the risk of cardiovasculardisease.

More than 40 percent of patients with myocardial infarction have lowHDL-C as a cardiac risk factor. (Genest, J. J., et al, Am. J. Cardiol.,1991, 67:1185-89). In the prospective and multicentric EuropeanConcerted Action on Thrombosis and Disabilities (ECAT) Angina PectorisStudy, Bolibar et al (Thromb. Haemost., 2000, 84:955-61) identified lowHDL-C and low apoA-I as the most important biochemical risk factors forcoronary events in patients with angiographically assessed CHD. Byconvention, the risk threshold value of HDL-C has been defined as 35mg/dL (0.9 mmol/L) in men and 45 mg/dL (1.15 mmol/L) in women [Expertpanel on detection, evaluation and treatment of high blood cholesterolin adults. The second report of the National Cholesterol EducationProgram (NCEP) expert panel on detection, evaluation and treatment ofhigh blood cholesterol in adults (Adult Treatment Panel II).Circulation. 1994; 89:1329-1445)]. Because of interaction, the strengthof the association between HDL-C and cardiovascular risk depends on thepresence of additional risk factors. Therefore, threshold values arehigher in men with diabetes mellitus or hypercholesterolemia or in thepresence of multiple risk factors (von Eckardstein A, and Assmann G.Curr Opin Lipidol. 2000; 11:627-637). Low HDL-C has been identified asthe most frequent familial dyslipoproteinemia in patients with prematuremyocardial infarction (Genest, J. J. Jr., Circulation. 1992;85:2025-2033). Finally, in the Helsinki Heart Study (Manninen, V. et al,Circulation. 1992; 85:37-45) and the High-Density-LipoproteinCholesterol Intervention Trial of the Department of Veterans Affairs(VA-HIT) study (Rubins, H. B. et al, N Engl J Med. 1999; 341:410-418),increases of HDL-C on treatment with gemfibrozil were correlated withthe prevention of CHD events. Thus, HDL-C has become an importantcomponent of algorithms to assess the global cardiovascular risk ofpatients and also a target for therapeutic intervention and for thedefinition of treatment goals.

Strategies to correct dyslipidemia in atherosclerosis generally involvediet and/or drugs. The threshold serum total cholesterol and LDLcholesterol concentrations above which diet and drug therapy should beinitiated, as well as the goals of therapy, have been defined by theNational Cholesterol Education Program (JAMA, 1993, 269:3015-23). Thetarget serum LDL-C is <160 mg/dl (4.3 mmol/1) for patients with no riskfactors or only one risk factor for CHD; <130 mg/dl (3.4 mmol/1) forpatients with 2 or more risk factors and less than 100 mg/dl (2.6mmol/1) for those with CHD. Persons with diabetes also fall into thethird category. A reasonable target for triglyceride concentration is200 mg/dl or less; higher values are associated with a doubling of therisk of cardiovascular disease when serum cholesterol concentrationexceeds 240 mg/dl or when the LDL-C/HDL-C ratio exceeds 5:1.

A number of studies have shown that reducing serum LDL-C below thetarget levels does not necessarily result in proportional reduction inthe risk of CHD [(The Scandinavian Simvastatin Survival Study Group.Randomized trial of cholesterol lowering in 4444 patients with coronaryheart disease, Lancet, 1994, 344:1383-89; Shepherd, J. et al, N. Engl.J. Med., 1995, 333:1301-7; Sachs, F. M. et al, N. Engl. J. Med., 1998,315:1001-9; Circulation, 1998, 97:1446-52; The West of Scotland CoronaryPrevention Study Group, Circulation, 1998, 97:1440-45; Pederson, T. R.,Circulation, 1998, 97:1453-60] because of the attenuation of thecholesterol-heart disease relation at lower serum cholesterolconcentrations (Grundy, S. M., Circulation, 1998, 97:1436-39).

Dietary treatment of hyperlipidemia is a necessary foundation for drugtreatment. Depending on the degree of hyperlipidemia, the Step I andStep II diets can be introduced sequentially. The Step II diet containsno more than 30% of calories from fat, less than 7% of calories fromsaturated fatty acids and less than 200 mg of cholesterol per day. Inlong term studies, the Step II diet decreased serum LDL-C concentrations8-15% (Knopp, R. H., et al, JAMA, 1997, 278:1509-15; Walden, C. E.,Arterioscl. Thromb. Vasc. Biol., 1997, 17:375-82; Denke, M. A., Arch.Intern. Med., 1995, 156:17-26). Diets more restricted in fat than theStep II diet result in little additional reduction in LDL-C, raise serumTG concentration and lower HDL-C.

The point to note, from the above, is that reducing LDL-C alone is oflittle value in reducing the risk of CHD. Further, diets meant forreducing LDL-C may reduce HDL-C to a similar degree (Hunninghake, D. B.et al, N. Engl. J. Med., 1993, 328:1213-19; Schaefer, E. J., et al,Arterioscl. Thromb. Vasc. Biol., 1995, 15:1079-85); Stefanick, M. L., N.Engl. J Med, 1998, 339:12-20).

Drug therapy is resorted to when the desired effects are not achievedwith diets alone. Statins are the most popular among the lipid loweringdrugs. These drugs lower serum LDL-C concentrations by upregulatingLDL-receptor activity as well as reducing the entry of LDL into thecirculation. The maximal reductions achieved with a statin ranges from24-60%. Statins also reduce the serum TG levels; but they are ofteninsufficient. Statins are ineffective in the treatment of patients withchylomicronemia. Adverse effects of statins include, gastrointestinalupset, muscle aches and hepatitis. Rarer problems include myopathy(muscle pain with serum creatine kinase concentrations more than 1,000 Uper litre), rashes, peripheral neuropathy, insomnia, bad or vivid dreamsand difficulty in sleeping or concentrating (Abramowica, M., Med Lett.,1996, 38:67-70; Vgontzas, A. N. et al, Clin. Pharmacol. Ther., 1991,50:730-37; Roth, T. et al, Clin. Cardiol., 1992, 15:426-32; Partinen, M.et al, Am. J. Cardiol., 1994, 73:876-80). Other lipid-lowering drugsinclude bile acid-binding resins (e.g., cholesteramine and colestipol),nicotinic acid, and fibrates.

Drug therapy is not recommended for premenopausal women and men under 35years of age unless they have serum LDL-C concentrations of more than220 mg/dl (5.7 mmol/1), because their immediate risk of heart disease islow [Summary of the second report of the National Cholesterol EducationProgram (NCEP): expert panel on detection, evaluation and treatment ofhigh blood cholesterol in adults, JAMA, 1993, 269:3015-23].

Thus, diets alone or in conjunction with lipid lowering drugs fail toyield the desired goal of safe lipid lowering. However, this goal isachievable with the present inventive composition containing the activeprinciples of seed of Emblica officinalis. Emblica has been in safe usein India for thousands of years as component of Ayurvedic preparations.The composition from seed of Emblica officinalis offers the twinbenefits of reducing the harmful LDL cholesterol and enhancing thedesirable HDL cholesterol.

A number of studies have shown that Emblica officinalis is useful forreducing total cholesterol, reducing triglyceride, reducing LDLcholesterol and enhancing HDL cholesterol.

Ritu Mathur et al show the hypolipidaemic effect of fruit juice ofEmblica officinalis in cholesterol fed rabbits. The juice is obtainedfrom deseeded Emblica officinalis. U.S. Pat. No. 6,124,268, Ghosaldiscloses a natural antioxidant composition from Emblica officinalisusing pericarp of fresh berries (Emblica officinalis). Biswas Gopa et alshow the hypolipidemic efficacy of Amla (Emblica officinalis). The Amlaused is dried Amla fruit juice powder. Muhammed et al evaluated theanti-hyperglycemic and lipid-lowering properties of Emblica officinalispowder in normal and diabetic human volunteers. Zhang et al disclosesthe phenolic constituents of Emblica officinalis juice. Chatterjee et aldiscloses a novel compounds with hypocholesteremic activity from crudeEmbilica officinialis (EO) fruit extracts. U.S. Pat. Nos. 7,780,996,8,158,167 and 8,455,020 discloses the method of reducing cholesterol,method of treating dyslipidemia and method of reducing triglyceride byextract of Emblica officinalis.

Amla is a fruit with wide range of medicinal properties. Our effort wasto find the most bioactive molecule/(s) or purified fraction havingbioactivity from Amla fruit. The fleshy part (pericarp) of Amla fruit isused for human consumption whereas Amla seeds are not edible anddiscarded. We evaluated different Amla extracts. Extracts prepared fromfresh fruit of Amla; fruit juice of whole Amla including the fleshy partand seeds of Amla; juice of fleshy part (pericarp); dried fruit; fleshof Amla fruit without seed; or Amla seed alone were evaluated for antihyperlipidemic activity. The methanol extract of all groups showedbeneficial activity, but the most unexpected and superior result wasobtained from the Amla seed alone extract. Amla seed alone extract wasable to significantly reduce the total cholesterol, LDL cholesterol,triglycerides, VLDL cholesterol and enhance the HDL cholesterol levels.Though Amla seed is not known to have any history of human consumption,we followed the lead with various extracts of Amla seed and found thatthe ethyl acetate portion of Amla seed extract is the most active. Theethyl acetate part was showing far superior activity compared to otherextracts with Amla seed and also against other extracts of Amla.

In view of the above, the disclosure provides a composition and methodof preparing an extract from the seed of Emblica officinalis unlikeother references where the extract is prepared from Emblica officinalis,especially from its fruits which found its application for the treatmentof reducing bad cholesterol, dyslipidemia and for reducing triglyceride.The disclosure provides a method of preparation of an extract of Emblicaofficinalis from the seed of Emblica officinalis and composition derivedcontain polyphenolic components and lipophilic components. The extractprepared from the seed of Emblica officinalis is useful for decreasingtotal cholesterol, decreasing triglyceride, reducing blood glucoselevel, enhancing HDL-Cholesterol level, increasing the HDL-Cholesterolto total cholesterol ratio, lowering LDL-Cholesterol level, reducing theCRP level, decreasing the intima media thickening, reducing atherogenicindex of plasma, increasing Apo A-1 levels, decreasing Apo B levels,decreasing Apo B to Apo A-1 ratio, reducing homocysteine level,reduction in glycosylated Hb, modulating TSH level, decreasing HMGCoAreductase activity, lowering total cholesterol without changing CoQ10levels even at a lower dosage level. The extract prepared from the seedof Emblica officinalis is useful for reducing hair fall in humans byapplying topically or by oral administration.

SUMMARY

The disclosure provides a medicinal composition of the extracts of seedof Emblica officinalis (Amla seed extract). The amla seed extractcomposition has applications as a nutraceutical or pharmaceuticalincluding for reducing the total cholesterol, reducing triglyceride,reducing blood glucose level, enhancing HDL-Cholesterol level,increasing the HDL-Cholesterol to total cholesterol ratio, loweringLDL-Cholesterol level, reducing the CRP level, decreasing the intimamedia thickening, reducing hair fall, reducing atherogenic index ofplasma, increasing Apo A-I levels, decreasing Apo B levels, decreasingApo B to Apo A-I ratio, reducing Homocysteine level, reduction inglycosylated Hb, modulating TSH level, decrease in HMGCoA reductaseactivity and lowering total cholesterol without a change in CoQ10 levelsin mammals especially human beings.

The composition of the extracts of seed of Emblica officinalis issuperior compared to extract from fruits of Emblica officinalis forindications including decreasing total cholesterol, decreasingtriglyceride, decreasing blood glucose level, enhancing HDL-Cholesterollevel, increasing the HDL-Cholesterol to total cholesterol ratio,lowering LDL-Cholesterol levels, decreasing CRP level, decreasing theintima media thickening, decreasing hair fall, reducing atherogenicindex of plasma, increasing Apo A-I levels, decreasing Apo B levels,decreasing Apo B to Apo A-I ratio, reducing Homocysteine level,reduction in glycosylated Hb, modulating TSH level, decreasing HMGCoAreductase activity and lowering total cholesterol without changing CoQ10levels. When same dosages of amla seed extract or amla fruit extractwere administered, amla seed extract showed superior results compared toamla fruit extract.

Even if the dosage of amla fruit extract was increased compared to amlaseed extract, amla seed extract administration showed superior resultscompared to amla fruit extract.

Some embodiments provide an amla seed blend composition (also referredto amla seed blend or Product 3). Product 3 is a blend of product 1 andproduct 2. Product 1 includes alpha linolenic acid, linoleic acid andoleic acid. Product 2 includes triterpenoids and hydroxycinnamic acids.In some embodiments, product 2 and product 1 are blended in a ratio ofProduct 2 to Product 1 ranging from about 1:60 to about 99:1.

The disclosure provides methods for preparing extracts of seed ofEmblica officinalis or blends of extracts of seeds of Emblicaofficinalis. In some embodiments, the disclosed extracts of seed ofEmblica officinalis or blends of extracts of seeds of Emblicaofficinalis can be used as a nutraceutical. In some embodiments, thedisclosed extracts of seed of Emblica officinalis or blends of extractsof seeds of Emblica officinalis can be used as a pharmaceutical. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis or blends of extracts of seeds of Emblica officinalisdecreased the total cholesterol level. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisdecreased triglyceride level. In some embodiments, administering thedisclosed extracts of seed of Emblica officinalis or blends of extractsof seeds of Emblica officinalis decreased blood glucose level. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis or blends of extracts of seeds of Emblica officinalisincreased level of HDL-Cholesterol. In some embodiments, administeringthe disclosed extracts of seed of Emblica officinalis or blends ofextracts of seeds of Emblica officinalis increased the HDL-Cholesterolto total cholesterol ratio. In some embodiments, administering thedisclosed extracts of seed of Emblica officinalis or blends of extractsof seeds of Emblica officinalis lowered LDL-Cholesterol level. In someembodiments, administering the disclosed seed extracts or blends ofextracts of seeds of Emblica officinalis decreased the CRP level. Insome embodiments, administering the disclosed extracts of seed ofEmblica officinalis decreased thickening of the intima media thickening.In some embodiments, administering the disclosed seed extracts or blendsof extracts of seed of Emblica officinalis decreased hair fall. In someembodiments, administering the disclosed seed extracts or blends ofextracts of seed of Emblica officinalis decreases the atherogenic indexof plasma. In some embodiments, administering the disclosed seedextracts or blends of extracts of seeds of Emblica officinalis increasedthe Apo A-I levels, decreased the Apo B levels and decreased the Apo Bto Apo A-I ratio. In some embodiments, administering the disclosed seedextracts or blends of extracts of seed of Emblica officinalis loweredHomocysteine level and glycosylated Hb level. In some embodiments,administering the disclosed seed extracts of seed or blends of extractsof seeds of Emblica officinalis modulated the TSH level. In someembodiments, administering the disclosed seed extracts or blends ofextracts of seed of Emblica officinalis decreased HMGCoA reductaseactivity. In some embodiments, administering the disclosed seed extractsor blends of extracts of seeds of Emblica officinalis lowered totalcholesterol without a change in CoQ10 levels.

In some embodiments, a method of producing the extract of seed ofEmblica offcinalisis disclosed. The method includes selecting the rawmaterial (fresh fruit of Emblica officinalis), followed by deseeding thefruits of Emblica officinalis. Then the seeds of Emblica officinalis arecrushed and extracted with solvents. Solvents include methanol, ethanol,isopropanol, n-butanol, methyl acetate, ethyl acetate, propyl acetate,n-butyl acetate and combinations thereof to obtain mixture. The mixtureis filtered. The filtrate is concentrated to obtain a concentratedextract. The concentrated extract is dried to form a dried extract. Thedried extract is macerated with water and partitioned with ethylacetate. The ethyl acetate part and aqueous part are formed and collectthe ethyl acetate part. Ethyl acetate part is concentrated and dried toform powder of ethyl acetate extract of seed of Emblica officinalis.

The extract of seed of Emblica officinalis can be prepared from fresh ordried seed of Emblica officinalis.

Some embodiments provide a blend of extracts of seeds of Emblicaofficinalis. The blend includes a first extract of seeds of Emblicaofficinalis having triterpenoids and hydroxycinnamic acids and a secondextract of seeds of Emblica officinalis having fatty acids, such as,alpha linolenic acid, linoleic acid and oleic acid. Methods of preparingblend of extracts of seeds of Emblica officinalis are provided. Methodsof treatment by administering blend of extracts of seeds of Emblicaofficinalis are provided.

The disclosure also provides a dosage form of the extract of seed ofEmblica officinalis. The disclosure provides a dosage form of an extractof seed of Emblica officinalis for oral administration. Dosage forms ofthe extract are selected from the group consisting of a capsule, tablet,granule, sachet, powder, paste, ointment, infusion, injection, ampoule,solution, suspension, emulsion, pills, oil, cream etc.

Further a dosage form of an extract of seed of Emblica officinalis isdisclosed for administering in a dosage ranging from about 5 mg to about500 mg to a human subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objectives and advantages of the disclosed teachings willbecome more apparent by describing in detail preferred embodimentsthereof with reference to the attached drawings in which:

FIG. 1: Flow chart depicting a method of preparation of ethyl acetateextract of methanol extract of seed of Emblica officinalis.

FIG. 2: Flow chart depicting a method of preparation of ethyl acetateextract of seed of Emblica officinalis.

FIG. 3: Flow chart depicting a method of preparation of pectinasetreated water extract of fruits of Emblica officinalis.

FIG. 4: Flow chart depicting a method of preparation of alcoholicextract of fruits of Emblica officinalis.

FIG. 5: Flow chart depicting a method of preparation of pectinasetreated water extract of fruits of Emblica officinalis without seed.

FIG. 6: Flow chart depicting a method of preparation of alcoholicextract of fruits of Emblica officinalis without seed.

FIG. 7: Flow chart depicting a method of preparation of powder of driedseed of Emblica officinalis.

FIG. 8: Flow chart depicting a method of preparation of powder of waterextract of dried seed of Emblica officinalis.

FIG. 9: Flow chart depicting a method of preparation of powder ofmethanol extract of dried seed of Emblica officinalis.

FIG. 10: Flow chart depicting a method of preparation of powder of fruitof Emblica officinalis.

FIG. 11: Flow chart depicting a method of preparation of powder of waterextract of fruit of Emblica officinalis.

FIG. 12: Flow chart depicting a method of preparation of powder ofmethanol extract of fruit of Emblica officinalis.

FIG. 13: Flow chart depicting a method of preparation of Amla seed blendcomposition.

FIGS. 14A and 14B: FIG. 14A provides a flow chart depicting a method ofpreparation of an extract of amla seed (first extract or sample 1). FIG.14B provides method of preparing second extract of amla seed (product 1)and method of blending sample 1 and product 1 to form an amla seed blendcomposition containing enriched triterpenoids and fatty acids.

DETAILED DESCRIPTION

The disclosure provides an extract of seed of Emblica officinalis. Thedisclosure also provides amla seed extract blend compositions. Thedisclosure relates to a medicinal composition of the extracts of seed ofEmblica officinalis with tripertenoids up to 70%.

The disclosure provides seed extracts of Emblica officinalis preparedfrom fresh or dry seeds of Emblica officinalis. In some embodiments, thedisclosed extracts of seed of Emblica officinalis can be used as anutraceutical. In some embodiments, the disclosed extracts of seed ofEmblica officinalis can be used as a pharmaceutical. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis decreased the total cholesterol level. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisdecreased triglyceride level. In some embodiments, administering thedisclosed extracts of seed of Emblica officinalis decreased bloodglucose level. In some embodiments, administering the disclosed extractsof seed of Emblica officinalis increased level of HDL-Cholesterol. Insome embodiments, administering the disclosed extracts of seed ofEmblica officinalis increased the HDL-Cholesterol to total cholesterolratio. In some embodiments, administering the disclosed extracts of seedof Emblica officinalis lowered LDL-Cholesterol level. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis decreased the CRP level. In some embodiments, administeringthe disclosed extracts of seed of Emblica officinalis decreasedthickening of the intima media thickening. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisdecreased hair fall. In some embodiments, administering the disclosedextracts of seed of Emblica officinalis decreases the atherogenic indexof plasma. In some embodiments, administering the disclosed extracts ofseed of Emblica officinalis increased the Apo A-1 levels, decreased theApo B levels and decreased the Apo B to Apo A-1 ratio. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis lowered Homocysteine level and glycosylated Hb level. Insome embodiments, administering the disclosed extracts of seed ofEmblica officinalis modulated the TSH level. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisdecreased HMGCoA reductase activity, lowering total cholesterol withouta change in CoQ10 levels.

In some embodiments, administering the disclosed extracts of seed ofEmblica officinalis promoted hair growth.

Atherogenic index of the plasma (AIP) is the logarithmically transformedratio of molar concentrations of triglycerides (TGs) to HDL-cholesterol(log (TG/HDL [mmol]). It has recently been proposed as a marker ofplasma atherogenecity because it is increased in people at higher riskfor coronary heart disease and is inversely correlated with LDL particlesize. The association of TGs and HDL-C in this simple ratiotheoretically reflects the balance between risk and protectivelipoprotein forces, and both TGs and HDL-C are widely measured andavailable. The strong correlation of AIP with lipoprotein particle sizemay explain its high predictive value. The Adult Treatment Panel III hasrecognized the important roles of HDL-C and TGs, calling thiscombination atherogenic dyslipidemia.

It has been suggest that AIP values of −0.3 to 0.1 are associated withlow, 0.1 to 0.24 with medium and above 0.24 with high CV risk. [Ref:Shabnam Niroumand, Mohammad Khajedaluee, Majid Khadem-Rezaiyan, MaryamAbrishami, Mohammadreza Juya, Gholamhasan Khodaee, and MalihehDadgarmoghaddam. Atherogenic Index of Plasma (AIP): A marker ofcardiovascular disease. Med J Islam Repub Iran. 2015; 29: 240.]. In ourstudy, the AIP has been reduced from 0.44 to 0.09 in amla seed extractgroup whereas in placebo group, there was very minor increase in AIP.This indicates the benefits of amla seed extract in managingcardiovascular health and reducing the risk of future cardio vasculardisease (CVD).

Apolipoprotein B (Apo B) is an important component of many lipoproteinsthat are involved in atherosclerosis and cardiovascular disease.Lipoproteins are the particles that transport cholesterol andtriglycerides in the blood stream. Lipoproteins are comprised ofproteins (apolipoproteins), phospholipids, triglycerides andcholesterol. The lipoproteins vary in the major lipoprotein present, andthe relative contents of the different lipid components. Apo B is animportant component of many of the most atherogenic lipoproteinparticles. ApoB occurs in 2 main forms, apo B 48 and apo B 100. Apo B 48is synthesized mainly by the small intestine. Apo B 100 is theapolipoprotein found in lipoproteins synthesized by the liver.Therefore, from the viewpoint of atherosclerosis and cardiovascularrisk, apoB100 is the important one. Apo B 48 is primarily found inchylomicrons. Apo B 100 is found in chylomicrons, VLDL, IDL and LDL. Allthese particles are atherogenic. Each of these particles contains asingle apoB molecule. Therefore, measurements of apo B represent thetotal burden of the main lipoprotein particles involved in theatherosclerotic process. Usually, 85-90 percent of apo B represent LDLparticles. Thus, apoB reflects particle concentration, similar to LDL.Apo B containing lipoproteins are the ones that are most likely to enterthe wall of the arteries. They are capable of trafficking cholesterolinto the artery wall, and if present in increased numbers they may bethe main initiating factor in atherosclerosis. Several studies haveshown that apoB may be a better predictor of cardiovascular disease riskthan LDL-C. The reference range of apoB levels in adults is less than130 mg/dL (1.3 g/L). Apo B levels are higher in males than in femalesand tend to increase with age.

In our study, the apo B decreased from 1.52 g/L to 1.11 g/L in amla seedextract group whereas in placebo group there was no change. Thisindicates the benefits of amla seed extract in reducing the risk of CVD.

Apo A-1 is a protein that has a specific role in the metabolism oflipids and is the main protein component in HDL, the “good cholesterol”.HDL removes excess cholesterol from cells and takes it to the liver forrecycling or disposal. Levels of Apo A-1 tend to rise and fall with HDLlevels, and deficiencies in Apo A-1 correlate with an increased risk ofdeveloping CVD. Low levels of Apo A-1 are associated with low levels ofHDL and impaired clearance of excess cholesterol from the body. Thereare some genetic disorders that lead to deficiencies in Apo A-1 (andtherefore to low levels of HDL). People with these disorders tend tohave abnormal lipid levels. Apolipoprotein A-1 (Apo-A1) is a structuraland functional protein that constitutes approximately 70% of the proteinin high density lipoprotein (HDL). The reference range of Apo-A1 variesby sex, as follows: Men: Greater than 120 mg/dL (1.2 g/L) and Women:Greater than 140 mg/dL (1.4 g/L). These levels decrease with age.

In our study, the Apo A-1 increased from 1.05 g/L to 1.32 g/L in amlaseed extract group whereas in placebo group there was no change. Thisindicates the benefits of amla seed extract in reducing the risk of CVD.

Low levels of Apo A-1, along with high concentrations of apo B, areassociated with an increased risk of cardiovascular disease. One studyfound that the apo B/Apo A1 ratio is more effective at predicting heartattack risk, than either the apo B or apoA1 measure alone. The normalvalues are 0.5 to 1.0.

In our study, the ratio of Apo B to Apo A-1 has been decreased from 1.45g/L to 0.84 g/L in amla seed extract group whereas in placebo groupthere was no change. This indicates the benefits of amla seed extract inreducing the risk of CVD. [Ref: Min Lu, Qun Lu, Yong Zhang, and GangTian. Apo B/Apo A1 is an effective predictor of coronary heart diseaserisk in overweight and obesity. J Biomed Res. 2011; 25(4): 266-273.]

Homocysteine is an amino acid and breakdown product of proteinmetabolism that, when present in high concentrations, has been linked toan increased risk of heart attacks and strokes. Amino acids are thebuilding blocks of proteins. When proteins break down, elevated levelsof amino acids like homocysteine may be found in the bloodstream. Havingelevated levels of homocysteine in the blood (hyperhomocysteinemia) isassociated with atherosclerosis and blood clots. Elevated homocysteinelevels are thought to contribute to plaque formation by damagingarterial walls. High levels may also act on blood platelets and increasethe risks of clot formation. Foods containing methionine are transformedinto homocysteine in the bloodstream. Homocysteine is converted in thebody to cysteine, with vitamin B6 facilitating this reaction.Homocysteine can also be recycled back into methionine using vitaminB12-related enzymes. If homocysteine cannot be converted into cysteineor returned to the methionine form, levels of homocysteine in the bodyincrease. Elevated homocysteine levels have been associated with heartattack, stroke, blood clot formation, and perhaps the development ofAlzheimer's disease.

Homocysteine levels are typically higher in men than women, and increasewith age. Common levels are in range of 10-15 μmol/L in plasma. In ourstudy, the homocystein level has been reduced from 23.58 to 15.61 μmol/Lin amla seed extract group whereas in placebo group, there was no changeas compared to baseline value. This indicates the benefits of amla seedextract in managing cardiovascular health and preventing the risk ofstroke, heart attack and other related risks. [Ref: Paul Ganguly andSreyoshi Fatima Alam. Role of homocysteine in the development ofcardiovascular disease. Nutr J. 2015; 14: 6.]

Coenzyme Q10 is the coenzyme for mitochondrial enzyme complexes involvedin oxidative phosphorylation in the production of ATP. That bioenergetic effect of CoQ10 is believed to be of fundamental importance inits clinical application, particularly as it relates to cells withexceedingly high metabolic demands such as cardiac myocytes. The secondfundamental property of CoQ10 involves its antioxidant (free radicalscavenging) functions. CoQ10 is the only known naturally occurring lipidsoluble antioxidant for which the body has enzyme systems capable ofregenerating the active reduced ubiquinol form. CoQ10 is carried in theblood with low density lipoprotein and serves to diminish the oxidationof LDL cholesterol in settings of oxidative stress. Other more recentlydiscovered aspects of CoQ10 function include its involvement inextramitochondrial electron transfer, e.g. plasma membraneoxidoreductase activity, involvement in cytosolic glycolysis, andpotential activity in both Golgi apparatus and lysosomes. CoQ10 alsoplays a role in improvement in membrane fluidity. CoQ10 is essential forall cellular ATP production and is of particular importance in heartmuscle function given that tissue's extreme energy requirements. Adeficiency of CoQ10 in the blood and the heart muscle has beendocumented in congestive heart failure. Primary and secondarydeficiencies of CoQ10 result in a number of neurologic and myopathicsyndromes. [Ref: Langsjoen P. H. and Langsjoen A. M. Overview of the Useof CoQ10 in Cardiovascular Disease. BioFactors 1999; 9(2-4): 273-284].

HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase) isthe rate-controlling enzyme of the mevalonate pathway, the metabolicpathway that produces cholesterol and other isoprenoids. Normally inmammalian cells this enzyme is suppressed by cholesterol derived fromthe internalization and degradation of low density lipoprotein (LDL) viathe LDL receptor as well as oxidized species of cholesterol. Competitiveinhibitors of the reductase induce the expression of LDL receptors inthe liver, which in turn increases the catabolism of plasma LDL andlowers the plasma concentration of cholesterol, an important determinantof atherosclerosis. This enzyme is thus the target of the widelyavailable cholesterol-lowering drugs known collectively as the statins.[Ref: DeBose-Boyd R A. Feedback Regulation of Cholesterol Synthesis:Sterol-Accelerated Ubiquitination and Degradation of HMG CoA Reductase.Cell Res. 2008; 18(6): 609-621.]

In the present study, amla seed extract has reduced the level of HMGCoAreductase, which is beneficial to reduce the cholesterol level. Thesynthetic HMGCoA reductase inhibitors (statins) also decrease thecholesterol level but at the same time, CoQ10 level also decreases whichis responsible for severe side effect. Amla seed extract reduced theHMGCoA level in dyslipidemia patients without affecting the CoQ10 levelafter treatment with Amla seed extract or placebo. This is veryimportant finding and unlike statins keeps Amla seed extract free fromsevere side effects associated with CoQ10 deficiency. In this study,highly significant reduction in HMGCoA reductase enzyme by Amla seedextract explains its mechanism of decreasing blood cholesterol andmanagement of cardio vascular health. At the same time, no change inCoQ10 level puts an advantage of Amla seed extract over statin therapyand makes it better choice than statins for treatment of dyslipidemia.

In an in vitro assay on HMG-CoA reductase inhibition, the amla seedextract inhibited the enzyme more than 98% at 200 μg/mL concentrationand IC50 value was calculated as 31.07. Low value of IC50 indicatesstrong inhibition of HMGCoA reductase enzyme and thus better forreduction of cholesterol. The IC50 for standard lovastatin was recordedas 29.7 which is very near to the IC50 of amla seed extract. Thus amlaseed extract is almost equally effective as standard lovastatin in theinhibiting of HMGCoA reductase in vitro. The water extract of dried amlaseed was less effective than amla seed extract and IC50 was found as142.7. [Ref: Holdgate, G. A., et. al., Molecular mechanism forinhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase byrosuvastatin. Biochem. Soc. Trans., 31, 528-531 (2003)].

Glycated hemoglobin (HbA1c) is a form of hemoglobin that is measuredprimarily to identify the three month average plasma glucoseconcentration. It is formed in a non-enzymatic glycation pathway byhemoglobin's exposure to plasma glucose. HbA1c is a measure of thebeta-N-1-deoxy fructosyl component of hemoglobin. Normal levels ofglucose produce a normal amount of glycated hemoglobin. As the averageamount of plasma glucose increases, the fraction of glycated hemoglobinincreases in a predictable way. This serves as a marker for averageblood glucose levels over the previous three months before themeasurement as this is the lifespan of red blood cells. Excessiveformation of early glycation products may adversely affect severalfunctions of blood vessels, lipid metabolism and prone to developdiabetic complications. In blood vessels, uptake of LDL may be enhanced;resulting in atherogenesis and also increases the free radical mediateddamage. These reversible biochemical abnormalities probably play a rolein the pathogenesis of the early functional changes in the diabeticmicrovasculature. Higher amounts of HbA1c in diabetic patients,indicating poorer control of blood glucose levels, have been associatedwith diabetic complications like; cardiovascular disease, nephropathy,and retinopathy.

HbA1c level below 5.7 percent is considered normal whereas between 5.7and 6.4 percent signals pre-diabetes. In our study, the level of HbA1chas been reduced from 6.7% to 5.59% in amla seed extract group whereasin placebo group, there was no change in HbA1c level. This indicates thebenefits of amla seed extract in managing the blood sugar level andpreventing diabetes.[Ref: Babbar A, Kaul N, Gupta S. Clinicalsignificance of glycosylated haemoglobin (HbA1C) over fasting bloodsugar for monitoring metabolic control in diabetic patients with orwithout complications. J Indian Med Assoc. 1996; 94(11):414-6.]

Thyroid function regulates a wide array of metabolic parameters. Thyroidfunction significantly affects lipoprotein metabolism as well as somecardiovascular disease (CVD) risk factors, thus influencing overall CVDrisk.

Thyroid hormones induce the 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase, which is the first step in cholesterolbiosynthesis. Moreover, triiodothyronine (T3) upregulates LDL receptorsby controlling the LDL receptor gene activation. This T3-mediated geneactivation is done by the direct binding of T3 to specific thyroidhormone responsive elements (TREs). Furthermore, T3 controls the sterolregulatory element-binding protein-2 (SREBP-2), which in turn regulatesLDL receptor's gene expression. T3 has also been associated withprotecting LDL from oxidation. Thyroid hormones can influence HDLmetabolism by increasing cholesteryl ester transfer protein (CETP)activity, which exchanges cholesteryl esters from HDL2 to the very lowdensity lipoproteins (VLDL) and TGs to the opposite direction. Inaddition, thyroid hormones stimulate the lipoprotein lipase (LPL), whichcatabolizes the TG-rich lipoproteins, and the hepatic lipase (HL), whichhydrolyzes HDL2 to HDL3 and contributes to the conversion ofintermediate-density lipoproteins (IDL) to LDL and in turn LDL to smalldense LDL.

Normal TSH level ranges from 0.4 to 4 μIU/ml. In our study, the amlaseed extract has increased the TSH level from 2.43 to 3.19, thusincreasing the lipoprotein lipase which reflects in decreasing the TGs.In placebo group there was slight decrease in TSH level. [Ref: KlempererJ D. Thyroid hormone and cardiac surgery. Thyroid. 2002; 12(6):517-21.]

The composition of the extracts of seed of Emblica officinalis issuperior compared to extract from fruits of Emblica officinalis forreducing the total cholesterol, reducing triglyceride, reducing bloodglucose level, enhancing HDL-Cholesterol level, increasing theHDL-Cholesterol to total cholesterol ratio, lowering LDL-Cholesterollevels, reducing the CRP level, decreasing the intima media thickening,reducing atherogenic index of plasma, increasing Apo A-1 levels,decreasing Apo B levels, decreasing Apo B to Apo A-1 ratio, reducingHomocysteine level, reduction in glycosylated Hb, modulating TSH level,decreasing HMGCoA reductase activity, lowering total cholesterol withoutchanging CoQ10 levels, reducing hair fall and promoting hair growth.

When same dosages of amla seed extract or amla fruit extract wereadministered, amla seed extract administration showed superior resultscompared to administering amla fruit extract.

Even if the dosage of amla fruit extract was increased compared to amlaseed extract, amla seed extract administration showed superior resultscompared to amla fruit extract.

Some embodiments provide an extract of seeds of Emblica officinalis. Theextract of Emblica officinalis includes triterpenoids, hydroxycinnamicacids, fatty acids and polyphenols. The triterpenoids includes beta sitosterol, beta amyrin and lupeol. The hydroxycinnamic acids includeferulic acid and p coumaric acid. The fatty acids include alphalinolenic acid, linoleic acid, oleic acid, stearic acid and palmiticacid. In some embodiments, the extract prepared from seed of Emblicaofficinalis includes polyphenolic components and lipophilic components.

In some embodiments of the extract of seeds of Emblica officinalis,triterpenoids ranges from about 0.5 to about 20% of the extract. In someembodiments, the extract of seeds of Emblica officinalis has about 0.5%to about of 5% of hydroxy cinnamic acids. In some embodiments, theextract of seeds of Emblica officinalis has about 25% to about 50% offatty acids. In some embodiments, the extract of seeds of Emblicaofficinalis has about 10% to about 95% of polyphenols. In someembodiments, the extract of seeds of Emblica officinalis has about 10%to about 20% of polyphenols. Some embodiments provide an extract ofseeds of Emblica officinalis having about 0.5 to about 20%triterpenoids, about 25% to about 50% fatty acids and about 10% to about20% polyphenol. The triterpenoids include beta sito sterol, beta amyrinand lupeol, the hydroxycinnamic acids include ferulic acid andp-coumaric acid, the fatty acids include alpha linolenic acid, linoleicacid, oleic acid, stearic acid and palmitic acid, and about 2.5% toabout 50% of the polyphenols are hydroxycinnamic acids.

In some embodiments, the extract of seeds of Emblica officinalis hasabout 9.5% triterpenoids. In some embodiments, the extract of seeds ofEmblica officinalis has about 4.3% of hydroxycinnamic acids. In someembodiments, the extract of seeds of Emblica officinalis has about 41.8%fatty acids. In some embodiments, the extract of seeds of Emblicaofficinalis has about 15% polyphenols.

In some embodiments of the extract of seeds of Emblica officinalis,triterpenoids enriched up to 70% of the extract. In some embodiments,the extract of seeds of Emblica officinalis has about 20% to about of70% of triterpenoids. In some embodiments, the extract of seeds ofEmblica officinalis has about 10% to about of 40% of hydroxy cinnamicacids. In some embodiments, the extract of seeds of Emblica officinalishas about 30% to about 60% of polyphenols.

In some embodiments, the extract of seeds of Emblica officinalis hasabout 63.5% triterpenoids. In some embodiments, the extract of seeds ofEmblica officinalis has about 27% of hydroxycinnamic acids.

Some embodiments provide a seed extract product having the extract ofseed of Emblica officinalis. The seed extract product includes fillerssuch as lactose, spray dried lactose, starch, dibasic calcium phosphate,tribasic calcium phosphate, microcrystalline cellulose, hydroxy propylmethyl cellulose, or calcium carbonate.

The disclosure relates to a composition having polyphenolic andlipophilic components obtained from extract of seed of Emblicaofficinalis wherein the polyphenolic components present in the extractof seed of Emblica officinalis ranges from 10% to 95%. Similarly theextract of seed of Emblica officinalis contains lipophilic componentsranges from 5% and above.

Some embodiments provide a method of preparing an extract of seeds ofEmblica officinalis. The method includes deseeding fresh fruits ofEmblica officinalis to obtain seeds of Emblica officinalis. The seedsare crushed. The crushed seeds are extracted with 95% methanol to obtaina residue and a supernatant. The supernatant is concentrated to obtain aconcentrated methanol extract. The concentrated methanol extract isdried to obtain a powder of methanol extract of seeds of Emblicaofficinalis. The method further includes macerating the powder ofmethanol extract of seeds of Emblica officinalis in water to obtain aliquid. The liquid is extracted with ethyl acetate to obtain an ethylacetate phase. The ethyl acetate phase is concentrated to obtain aconcentrated ethyl acetate phase. The concentrated ethyl acetate phaseis dried to obtain a powder of ethyl acetate extract of methanol extractof seed of Emblica officinalis.

Powder of ethyl acetate extract of methanol extract of seed of Emblicaofficinalis is blended with microcrystalline cellulose to obtain a blendof powder of ethyl acetate extract of methanol extract of seed ofEmblica officinalis and microcrystalline cellulose.

Some embodiments provide a method of preparing an extract of seeds ofEmblica officinalis. The method includes deseeding fresh fruits ofEmblica officinalis to obtain seeds of Emblica officinalis. The seedsare crushed. The crushed seeds are extracted with ethyl acetate toobtain a supernatant. The supernatant is concentrated to obtain aconcentrated ethyl acetate extract. The concentrated ethyl acetateextract is dried to obtain a powder of ethyl acetate extract of seeds ofEmblica officinalis.

In some embodiments, the fresh fruit of Emblica officinalis is cleanedand deseeded. Seeds are crushed and extracted for about 1 hr usingmethanol in an extractor with reflux condenser to obtain residue andsupernatant. The residue and supernatant is separated by draining outthe supernatant from the extractor bottom through the filter cloth. Theresultant supernatant is concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form concentratedextract. Later the concentrated extract is dried under vacuum at above500 mm of mercury to form powder of methanol extract of seed of EmblicaOfficinalis.

The powder of methanol extract of seed of Emblica officinalis ismacerated with water and partitioned with ethyl acetate. Collect theethyl acetate part. Concentrate the ethyl acetate part in an Agitatedthin film evaporator and dried under vacuum at above 500 mm of mercuryto form powder of ethyl acetate extract of methanol extract of seed ofEmblica officinalis. Method of preparing the extract is provided in FIG.1.

In one embodiment fresh fruit of Emblica officinalis is cleaned anddeseeded. Seeds are crushed and extracted for 5 hrs using ethyl acetateat 78° C. in a Soxhlet extractor and then filtered. The resultantextract is concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 75° C. to form concentrated extract. Later theconcentrated extract is dried under vacuum at above 500 mm of mercury toform powder of ethyl acetate extract of seed of Emblica officinalis.Method of preparing the extract is provided in FIG. 2.

In another embodiment the method of manufacture of a powder of apectinase treated water extract of fruits of Emblica officinalis is bypulping fruits of Emblica officinalis with demineralized water to createslurry. The slurry is treated with pectinase and then filtered to obtaina solution. The solution is concentrated and dried under vacuum. Driedproduct is pulverized and sieved through 30 mesh to obtain a powder ofthe pectinase treated water extract of fruits of Emblica officinalis.Method of preparing the extract is provided in FIG. 3.

Some embodiments disclose a method of preparing a powder of an alcoholicextract of fruits of Emblica officinalis. Fresh fruits of Emblicaofficinalis are pulverized and extracted for about 1 hr using 95%methanol in an extractor with reflux condenser to obtain residue andsupernatant. The residue and supernatant is separated by draining outthe supernatant from the extractor bottom through the filter cloth. Theresultant supernatant is concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form concentratedextract. Later the concentrated extract is dried under vacuum at above500 mm of mercury to form powder of methanol extract of fruit of EmblicaOfficinalis. Method of preparing the extract is provided in FIG. 4.

In some embodiments, a method of manufacture of a powder of a pectinasetreated water extract of fruits of deseeded Emblica officinalis isdisclosed. Fruit of deseeded Emblica officinalis is made into a pulpalong with demineralized water to create slurry. The slurry is treatedwith pectinase and then filtered to obtain a solution. The solution isconcentrated and dried under vacuum. The dried material is pulverizedand sieved through 30 mesh to obtain a powder of the pectinase treatedwater extract of fruits of deseeded Emblica officinalis. Method ofpreparing the extract is provided in FIG. 5.

In another embodiment, a method of preparing a powder of an alcoholicextract of fruits of deseeded Emblica officinalis is disclosed. Freshfruits of Emblica officinalis are deseeded and deseeded fruits arepulverized and extracted for about 1 hr using 95% methanol in anextractor with reflux condenser to obtain residue and supernatant. Theresidue and supernatant is separated by draining out the supernatantfrom the extractor bottom through the filter cloth. The resultantsupernatant is concentrated in an Agitated thin film evaporator (ATFE)at a temperature of 65° C. to form concentrated extract. Later theconcentrated extract is dried under vacuum at above 500 mm of mercury toform powder of methanol extract of fruit of deseeded EmblicaOfficinalis. Method of preparing the extract is provided in FIG. 6.

Some embodiments provide a composition having the extract of seeds ofEmblica officinalis. Some embodiments provide a method of treatment byadministering to a human subject about 5 mg to about 500 mg of theextract of seeds of Emblica officinalis. Some embodiments provide adosage form having the extract of seeds of Emblica officinalis. Thedosage form includes a dosage of the extract of seeds of Emblicaofficinalis ranging from about 5 mg to about 500 mg. Some embodiments ofthe method administer a dose of about 5 mg to about 500 mg per day to ahuman. Some embodiments administer a dose of about 5 mg to about 500 mgtwo or three times per day to a human. In some embodiments, the extractof seed of Emblica officinalis is administered in a dosage of 5 mg to100 mg in humans. The dosage form is administered in single or multipledoses per day. Some embodiments provide a dosage form such as a capsule,tablet, granule, sachet, powder, paste, ointment, infusion, injection,ampoule, solution, suspension, emulsion, pills, oil, or, cream.

Some embodiments provide a method of reducing total cholesterol byadministering an extract of seeds of Emblica officinalis. Someembodiments provide a method of reducing triglyceride by administeringan extract of seeds of Emblica officinalis. Some embodiments provide amethod of reducing blood glucose level by administering an extract ofseeds of Emblica officinalis. Some embodiments provide a method ofenhancing HDL-Cholesterol by administering an extract of seeds ofEmblica officinalis. Some embodiments provide a method of increasing aratio of HDL cholesterol to total cholesterol by administering anextract of seeds of Emblica officinalis. Some embodiments provide amethod of lowering LDL-Cholesterol levels by administering an extract ofseeds of Emblica officinalis. Some embodiments provide a method oflowering VLDL by administering an extract of seeds of Emblicaofficinalis. Some embodiments provide a method of reducing CRP level byadministering an extract of seeds of Emblica officinalis. Someembodiments provide a method of decreasing the intima media thickeningby administering an extract of seeds of Emblica officinalis. Someembodiments provide a method of reducing hair fall by administering anextract of seeds of Emblica officinalis. Some embodiments provide amethod of reducing atherogenic index of plasma by administering anextract of seeds of Emblica officinalis. Some embodiments provide amethod of increasing Apo A-1 levels, method of decreasing Apo B levelsand method of decreasing the Apo B to Apo A-1 ratio by administering anextract of seeds of Emblica officinalis. Some embodiments provide amethod of lowering homocysteine level and glycosylated Hb level byadministering an extract of seeds of Emblica officinalis. Someembodiments provide a method to modulate the TSH level by administeringan extract of seeds of Emblica officinalis. Some embodiments provide amethod of lowering HMGCoA reductase activity by administering an extractof seeds of Emblica officinalis. Some embodiments provide a method oflowering total cholesterol without changing CoQ10 levels byadministering an extract of seeds of Emblica officinalis. Someembodiments provide a method of decreasing risk for coronary heartdisease by administering an extract of seeds of Emblica officinalis.Some embodiments provide a method of decreasing atherogenic dyslipidemiaby administering an extract of seeds of Emblica officinalis.

Some embodiments provide an amla seed blend composition. Someembodiments of the amla seed blend composition are referred to asproduct 3. The amla seed blend composition is a blend of varying ratiosof product 1 and product 2. Product 1 includes alpha linolenic acid,linoleic acid and oleic acid. Product 2 includes triterpenoids andhydroxycinnamic acids. Product 2 includes triterpenoids and polyphenols.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 ranging from about 1:1 to about 99:1. In someembodiments, product 2 and product 1 are blended in a ratio of product 2to product 1 ranging from about 1:60 to about 99:1. In some embodiments,product 2 and product 1 are blended in a ratio of product 2 to product 1ranging from about 1:1 to about 1:10. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 2:3.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 of about 1:2. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 1:1.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 of about 3:2. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 10:1or 90:9. In some embodiments, product 2 and product 1 are blended in aratio of product 2 to product 1 of about 95:5 or 19:1. In someembodiments, product 2 and product 1 are blended in a ratio of product 2to product 1 of about 3:1 or 75:25. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 1:5.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 of about 1:10. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 1:3.

In some embodiments, the blending of product 2 and product 1 provides anamla seed blend composition having about 6% to about 50% oftriterpenoids. The triterpenoids include among others beta-sitosterol,beta amyrin and lupeol. In some embodiments, the blending of product 2and product 1 provides an amla seed product having about 2% to about 20%of hydroxycinnamic acids. The hydroxycinnamic acids include ferulic acidand p-coumaric acid. In some embodiments, combining product 2 andproduct 1 results in an amla seed product having about 10% to about 60%of fatty acids. The fatty acids include unsaturated and saturated fattyacids. The unsaturated fatty acids include alpha linolenic acid,linoleic acid and oleic acid.

Some embodiments provide an amla seed blend composition as a blend ofvarying ratios of product 1 (extract from Example 13) and product 2(extract from Example 13). Product 2 includes triterpenoids andhydroxycinnamic acids. Product 1 includes alpha linolenic acid, linoleicacid and oleic acid. In some embodiments, product 2 and product 1 areblended in a ratio of product 2 to product 1 ranging from about 1:60 toabout 99:1. Table A provides how blending of product 2 and product 1 in1:5 ratio results in a composition having about triterpenoids 8.3%,hydroxycinnamic acid 4.8% and fatty acids 64.6%. Blending of product 2and product 1 in 1:2 ratio provides an amla seed blend compositionhaving about triterpenoids 16%, hydroxycinnamic acid 9.8% and fattyacids 51.6%. Blending of product 2 and product 1 in 1:1 ratio providesan amla seed blend composition having about triterpenoids 25%,hydroxycinnamic acid 14.65% and fatty acids 38.8%. Blending of product 2and product 1 in 3:2 ratio provides an amla seed blend compositionhaving about triterpenoids 30%, hydroxycinnamic acid 17.6% and fattyacids 31%. Blending of product 2 and product 1 in 2:1 ratio provides anamla seed blend composition having about triterpenoids 33.3%,hydroxycinnamic acid 19.5% and fatty acids 25.8%. Blending of product 2and product 1 in 3:1 ratio provides an amla seed blend compositionhaving about triterpenoids 37.5%, hydroxycinnamic acid 22.1% and fattyacids 19.4%. Blending of product 2 and product 1 in 5:1 ratio providesan amla seed blend composition having about triterpenoids 41.6%,hydroxycinnamic acid 24.6% and fatty acids 12.9%. Blending of product 2and product 1 in 10:1 ratio provides an amla seed blend compositionhaving about triterpenoids 45%, hydroxycinnamic acid 26.6% and fattyacids 7.1%. Blending of product 2 and product 1 in 20:1 ratio providesan amla seed blend composition having about triterpenoids 47.6%,hydroxycinnamic acid 27.9% and fatty acids 3.69%. Blending of product 2and product 1 in 2:3 ratio provides an amla seed blend compositionhaving about triterpenoids 20%, hydroxycinnamic acid 11.7% and fattyacids 46.2%.

Some embodiments provide a dosage form having the amla seed blendcomposition, which is a blend of product 2 and product 1 in 1:5, 1:2,2:3, 1:1, 3:2, 2:1, 3:1, 5:1, 10:1 and 20:1 ratios of product 2 toproduct 1 but not limited to the above ratios. Some embodiments providea dosage form having amla seed blend composition and a filler. In someembodiments, amla seed blend composition and filler are blended in aratio of amla seed blend composition to filler ranging from about 10:1to about 3:1. In some embodiments, amla seed blend composition andfiller are blended in a ratio of extract of seeds of Emblica officinalisto filler of about 4.5:1.

In some embodiments, administering theamla seed blend composition whichis a blend of product 2 and product 1 in 1:5, 1:2, 2:3, 1:1, 3:2, 2:1,3:1, 5:1, 10:1 and 20:1 ratios of product 2 to product 1 but not limitedto the above ratios, is useful for decreasing total cholesterol,decreasing triglyceride, reducing blood glucose level, enhancingHDL-Cholesterol level, increasing the HDL-Cholesterol to totalcholesterol ratio, lowering LDL-Cholesterol level, reducing the CRPlevel, decreasing the intima media thickening, reducing atherogenicindex of plasma, increasing Apo A-1 levels, decreasing Apo B levels,decreasing Apo B to Apo A-1 ratio, reducing Homocysteine level,reduction in glycosylated Hb, modulating TSH level, decreasing HMGCoAreductase activity, lowering total cholesterol without changing CoQ10levels even at a lower dosage level.

TABLE A Amla seed blend composition of product 2 and product 1 indifferent ratios. Product 2 Product 1 Ratios TriterpenoidsHydroxycinnamic acid Fatty acids product 2:product 1 (%) (%) (%) 1:5 8.34.8 64.6 1:2 16 9.8 51.6 2:3 (product 3) 20 11.7 46.2 1:1 25 14.7 38.83:2 30 17.6 31 2:1 33.3 19.5 25.8 3:1 37.5 22.1 19.4 5:1 41.6 24.6 12.910:1  45 26.6 7.1 20:1  47.6 27.9 3.69

Some embodiments provide a method of preparing an amla seed blend havingproduct 1 and the product 2. Some embodiments provide a method ofpreparing the product 1. Fresh fruits of Emblica officinalis aredeseeded to obtain seeds of Emblica officinalis. The seeds are crushed.The crushed seeds are extracted with 95% methanol to obtain a residueand a supernatant. The supernatant is concentrated resulting in aconcentrated methanol extract. The concentrated methanol extract isdried to obtain a powder of methanol extract of seeds of Emblicaofficinalis. The powder of methanol extract of seeds of Emblicaofficinalis is dispersed in water to obtain a dispersion. The dispersionis extracted with hexane in a liquid-liquid extractor following which ahexane and a water phase is collected. The hexane phase is concentratedto obtain a liquid form of a concentrated hexane extract. The liquidform of the concentrated hexane extract is cooled, whereby obtainprecipitates or crystals are formed and a liquid portion is obtained.The liquid portion is separated from the precipitates or crystals toobtain a liquid product 1. Some embodiments provide a method ofpreparing product 2 from Amla seed. The method includes extracting thewater phase collected above after hexane extraction with ethyl acetateto obtain an ethyl acetate phase. The ethyl acetate phase isconcentrated to obtain a concentrated ethyl acetate phase. Theconcentrated ethyl acetate phase is dried to obtain a powder of an ethylacetate extract. The powder of ethyl acetate extract is mixed with waterto obtain a liquid of powder of ethyl acetate extract. The liquid ofpowder of ethyl acetate extract is loaded onto an ion exchange column.The ion exchange column is eluted with water to obtain a water fraction(also referred to as Fraction 1). Next, the ion exchange column iseluted with 50% methanol to obtain a Fraction 2. Then the ion exchangecolumn is eluted with 80% methanol to obtain a Fraction 3. Fraction 1 isconcentrated to obtain a concentrate of Fraction 1. The concentrate ofFraction 1 is dried to obtain a powder of Fraction 1. Fraction 2 isconcentrated to obtain a concentrate of Fraction 2. The concentrate ofFraction 2 is dried to obtain a powder of Fraction 2. Fraction 3 isconcentrated to obtain a concentrate of Fraction 3. The concentrate ofFraction 3 is dried to obtain a powder of Fraction 3. The powder ofFraction 1, the powder of Fraction 2 and the powder of Fraction 3 arecombined to obtain Product 2. Some embodiments of Product 2 have thepowder of Fraction 1, the powder of Fraction 2 and the powder ofFraction 3 in a 0.5:1:0.75 ratio of Fraction 1:Fraction 2:Fraction 3.Product 1 includes alpha linolenic acid, linoleic acid and oleic acid.Product 2 includes triterpenoids and hydroxycinnamic acids. In someembodiments, product 2 and product 1 are blended in a ratio of product 2to product 1 ranging from about 1:1 to about 99:1. In some embodiments,product 2 and product 1 are blended in a ratio of product 2 to product 1ranging from about 1:60 to about 99:1. In some embodiments, product 2and product 1 are blended in a ratio of product 2 to product 1 rangingfrom about 10:1 to about 1:10. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 2:3.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 of about 1:2. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 1:1.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 of about 3:2. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 10:1or 90:9. In some embodiments, product 2 and product 1 are blended in aratio of product 2 to product 1 of about 95:5 or 19:1. In someembodiments, product 2 and product 1 are blended in a ratio of product 2to product 1 of about 3:1 or 75:25. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 1:5.In some embodiments, product 2 and product 1 are blended in a ratio ofproduct 2 to product 1 of about 1:10. In some embodiments, product 2 andproduct 1 are blended in a ratio of product 2 to product 1 of about 1:3.Method of preparing the extract is provided in FIG. 13.

Some embodiments provide a dosage form having the amla seed blendcomposition, which is a blend of product 1 and product 2. The dosageforms include a dosage of the amla seed blend composition ranging fromabout 5 mg to about 500 mg per dose in human subject. The dosage form isadministered in single or multiple doses per day. Some embodimentsprovide a dosage form such as a capsule, tablet, granule, sachet,powder, paste, ointment, infusion, injection, ampoule, solution,suspension, emulsion, pills, oil, or, cream.

Some embodiments provide an amla seed blend composition, which is ablend of product 1 and product 2. Some embodiments provide a method ofreducing total cholesterol by administering the amla seed blendcomposition. Some embodiments provide a method of reducing triglycerideby administering the amla seed blend composition. Some embodimentsprovide a method of reducing blood glucose level by administering anamla seed blend composition. Some embodiments provide a method ofenhancing HDL-Cholesterol by administering an amla seed blendcomposition. Some embodiments provide a method of increasing a ratio ofHDL cholesterol to total cholesterol by administering an amla seed blendcomposition. Some embodiments provide a method of loweringLDL-Cholesterol levels by administering an amla seed blend composition.Some embodiments provide a method of lowering VLDL by administering anamla seed blend composition. Some embodiments provide a method ofreducing CRP level by administering an amla seed blend composition. Someembodiments provide a method of decreasing the intima media thickeningby administering an amla seed blend composition. Some embodimentsprovide a method of reducing hair fall by administering an amla seedblend composition. Some embodiments provide a method of reducingatherogenic index of plasma by administering an amla seed blendcomposition having Product 1 and Product 2. Some embodiments provide amethod of increasing Apo A-1 levels, method of decreasing Apo B levelsand method of decreasing the Apo B to Apo A-1 ratio by administering anamla seed blend composition. Some embodiments provide a method oflowering homocysteine level and glycosylated Hb level by administeringan amla seed blend composition. Some embodiments provide a method tomodulate TSH level by administering an amla seed blend composition. Someembodiments provide a method of lowering HMGCoA reductase activity byadministering an amla seed blend composition. Some embodiments provide amethod of lowering total cholesterol without a change in CoQ10 levels byadministering an amla seed blend composition.

Some embodiments provide a first extract of seeds of Emblica officinalishaving about 20% to about 70% triterpenoids; and, about 10% to about 40%hydroxycinnamic acids.

Some embodiments provide a blend of extracts of seeds of Emblicaofficinalis having the first extract of seeds of Emblica officinalis(some embodiments are provided in Example 27), and, a second extract ofEmblica Officinalis (some embodiments are provided in Example 13). Inthe blend of extracts of Emblica officinalis, a weight ratio of thefirst extract of Emblica Officinalis to the second extract of Emblicaofficinalis in the blend ranges from about 1:60 to about 99:1. Thesecond extract of Emblica officinalis includes fatty acids, such as,alpha linolenic acid, linoleic acid and oleic acid. In some embodimentsof the blend of extracts of seeds of Emblica officinalis, the weightratio of the first extract of seeds of Emblica Officinalis to the secondextract of Emblica officinalis in the blend is about 1:1. Someembodiments of the blend of extracts of seeds of Emblica officinalisinclude: about 10% to about 50% triterpenoids; about 3% to about 30%hydroxycinnamic acids, and, about 10% to about 60% of second extract ofseeds of Emblica officinalis. In some embodiments, the blending ofsample 1 and product 1 provides an amla seed blend composition havingabout 10% to about 50% of triterpenoids. The triterpenoids include amongothers beta-sitosterol, beta amyrin and lupeol. The hydroxycinnamicacids include ferulic acid and p-coumaric acid. In some embodiments,combining sample 1 and product 1 results in an amla seed product havingabout 10% to about 60% of fatty acids. The fatty acids includeunsaturated and saturated fatty acids. The unsaturated fatty acidsinclude alpha linolenic acid, linoleic acid and oleic acid.

Some embodiments provide an extract of seeds of Emblica officinalishaving about 0.5% to about 20% triterpenoids, about 0.5% to about 5% ofhydroxycinnamic acids, and, about 25% to about 50% fatty acids. In someembodiments, the extract of seeds of Emblica officinalis has about 9.5%triterpernoids, about 4.3% hydroxycinnamic acids and about 41.8% fattyacids. Some embodiments provide a dosage form having the extract and afiller. The filler can be lactose, spray dried lactose, starch, dibasiccalcium phosphate, tribasic calcium phosphate, microcrystallinecellulose, hydroxy propyl methyl cellulose, calcium carbonate, orcombinations thereof. The dosage form can be capsule, tablet, granule,sachet, powder, paste, ointment, infusion, injection, ampoule, solution,suspension, emulsion, pills, oil, or, cream. Some embodiments provide amethod of decreasing atherogenic index of plasma in a mammal byadministering the extract of seeds of Emblica officinalis. Theatherogenic index of plasma is a logarithmic ratio of triglycerides toHDL cholesterol. Some embodiments include methods of use of extracts orblends or compositions of Emblica officinalis by administering a dosageof extract ranging from about 6 mg to about 500 mg daily for 12 weeks.Other dosages administered include administering about 250 mg to about500 mg of extracts of Emblica officinalis, blend of Emblica officinalisor composition having extract or blend of Emblica officinalis. Dosagescan be administered about two to three times per day for a duration of12 weeks resulting in the physiological or treatment effects of thedisclosed extracts, blends or compositions. Some embodiments provide amethod of decreasing risk for coronary heart disease by administeringthe extract of seeds of Emblica officinalis. Some embodiments provide amethod of decreasing atherogenic dyslipidemia administering the extractof seeds of Emblica officinalis. Some embodiments provide a method ofdecreasing ratio of Apo B to Apo A1 by administering the extract ofseeds of Emblica officinalis. Some embodiments provide a method oflowering homocysteine level by administering the extract of seeds ofEmblica officinalis. Some embodiments provide a method of loweringglycosylated hemoglobin level by administering the extract of seeds ofEmblica officinalis. Some embodiments provide a method of modulating TSHlevels by administering the extract of seeds of Emblica officinalis.Some embodiments provide a method of lowering HMGCoA reductase byadministering the extract of seeds of Emblica officinalis. Someembodiments provide a method of lowering total cholesterol withoutaltering CoQ10 level by administering the extract of seeds of Emblicaofficinalis.

Some embodiments provide a dosage form of the extract of seeds ofEmblica officinalis and a filler. In some embodiments, extract of seedsof Emblica officinalis and filler are blended in a ratio of extract ofseeds of Emblica officinalis to filler ranging from about 10:1 to about3:1. In some embodiments, extract of seeds of Emblica officinalis andfiller are blended in a ratio of extract of seeds of Emblica officinalisto filler of about 5:1. In some embodiments, extract of seeds of Emblicaofficinalis and filler are blended in a ratio of extract of seeds ofEmblica officinalis to filler of about 6:1. In some embodiments, extractof seeds of Emblica officinalis and filler are blended in a ratio ofextract of seeds of Emblica officinalis to filler of about 4.5:1.

In another embodiment, 500 mg capsule having extract of seeds of Emblicaofficinalis and filler includes a first extract to filler ratio of about10:1 to about 3:1. Some embodiments have a ratio extract of seeds ofEmblica officinalis to fillers of about 4.5:1. A 500 mg capsule ofextract of seeds of Emblica officinalis with filler has about 400 mg toabout 440 mg of extract of seeds of Emblica officinalis. Someembodiments have about 430 mg of the extract of seeds of Emblicaofficinalis. Some embodiments have about 410 mg of extract of seeds ofEmblica officinalis. A 500 mg capsule of extract of seeds of Emblicaofficinalis with filler can have about 60 mg to about 100 mg of filler.Some embodiments have about 90 mg filler. A 500 mg capsule of extract ofseeds of Emblica officinalis and filler can have triterpenoids rangingfrom about 25 mg to about 40 mg, hydroxycinnamic acid ranging from about10 mg to about 20 mg and fatty acids ranging from about 130 mg to about150 mg. In some embodiments of the dosage form, the triterpenoids can beabout 31.98 mg, hydroxycinnamic acid about 14.35 mg and fatty acid about140.6 mg.

Some embodiments of the amla seed blend composition are referred to asSample 2. Sample 2 is a blend of varying ratios of sample 1 andproduct 1. Product 1 includes alpha linolenic acid, linoleic acid andoleic acid. Sample 1 includes triterpenoids and hydroxycinnamic acids.In some embodiments, sample 1 and product 1 are blended in a ratio ofsample 1 to product 1 ranging from about 1:1 to about 99:1. In someembodiments, sample 1 and product 1 are blended in a ratio of sample 1to product 1 ranging from about 1:60 to about 99:1. In some embodiments,sample 1 and product 1 are blended in a ratio of sample 1 to product 1ranging from about 1:1 to about 1:10. In some embodiments, sample 1 andproduct 1 are blended in a ratio of sample 1 to product 1 of about 2:3.In some embodiments, sample 1 and product 1 are blended in a ratio ofsample 1 to product 1 of about 1:2. In some embodiments, sample 1 andproduct 1 are blended in a ratio of sample 1 to product 1 of about 1:1.In some embodiments, sample 1 and product 1 are blended in a ratio ofsample 1 to product 1 of about 3:2. In some embodiments, sample 1 andproduct 1 are blended in a ratio of sample 1 to product 1 of about 10:1or 90:9. In some embodiments, sample 1 and product 1 are blended in aratio of sample 1 to product 1 of about 95:5 or 19:1. In someembodiments, sample 1 and product 1 are blended in a ratio of sample 1to product 1 of about 3:1 or 75:25. In some embodiments, sample 1 andproduct 1 are blended in a ratio of sample 1 to product 1 of about 1:5.In some embodiments, sample 1 and product 1 are blended in a ratio ofsample 1 to product 1 of about 1:10. In some embodiments, sample 1 andproduct 1 are blended in a ratio of sample 1 to product 1 of about 1:3.

Some embodiments provide an amla seed blend composition. The amla seedblend composition is a blend of varying ratios of sample 1 (extract fromExample 27) and product 1 (extract from Example 13). Sample 2 includestriterpenoids and hydroxycinnamic acids. Product 1 includes alphalinolenic acid, linoleic acid and oleic acid. In some embodiments,sample 1 and product 1 are blended in a ratio of sample 1 to product 1ranging from about 1:60 to about 99:1. Table B provides how blending ofsample 1 and product 1 in 1:5 ratio provides an amla seed blendcomposition having about triterpenoids 10.6%, hydroxycinnamic acid 4.5%and fatty acids 64.6%. Blending of sample 1 and product 1 in 1:2 ratioprovides an amla seed blend composition having about triterpenoids21.2%, hydroxycinnamic acid 9% and fatty acids 51.6%. Blending of sample1 and product 1 in 2:3 ratio provides an amla seed blend compositionhaving about triterpenoids 25.4%, hydroxycinnamic acid 10.8% and fattyacids 46.2%. Blending of sample 1 and product 1 in 3:2 ratio provides anamla seed blend composition having about triterpenoids 38.1%,hydroxycinnamic acid 16.2% and fatty acids 31%. Blending of sample 1 andproduct 1 in 2:1 ratio provides an amla seed blend composition havingabout triterpenoids 42.3%, hydroxycinnamic acid 18% and fatty acids25.8%. Blending of sample 1 and Product 1 in 3:1 ratio provides an amlaseed blend composition having about triterpenoids 37.5%, hydroxycinnamicacid 22.1% and fatty acids 19.4%. Blending of Sample 1 and product 1 in5:1 ratio provides an amla seed blend composition having abouttriterpenoids 52.9%, hydroxycinnamic acid 22.5% and fatty acids 12.9%.Blending of sample land product 1 in 10:1 ratio provides an amla seedblend composition having about triterpenoids 57.7%, hydroxycinnamic acid24.5% and fatty acids 7.1%. Blending of sample 1 and product 1 in 20:1ratio provides an amla seed blend composition having about triterpenoids60.5%, hydroxycinnamic acid 25.7% and fatty acids 3.7%. Blending ofsample 1 and product 1 in 1:1 ratio provides an amla seed blendcomposition having about triterpenoids 31.8%, hydroxycinnamic acid 13.5%and fatty acids 38.8%.

Some embodiments provide a dosage form having the amla seed blendcomposition, which is a blend of sample 1 and product 1 in 1:5, 1:2,2:3, 1:1, 3:2, 2:1, 3:1, 5:1, 10:1 and 20:1 ratios of sample 1 toproduct 1 but not limited to the above ratios. Some embodiments providea dosage form having amla seed blend composition and a filler. In someembodiments, amla seed blend composition and filler are blended in aratio of amla seed blend composition to filler ranging from about 10:1to about 3:1. In some embodiments, amla seed blend composition andfiller are blended in a ratio of extract of seeds of Emblica officinalisto filler of about 4.5:1.

Sample 1 and product 1 in 1:5, 1:2, 2:3, 1:1, 3:2, 2:1, 3:1, 5:1, 10:1and 20:1 ratios of sample 1 to product 1 but not limited to the aboveratios, is useful for decreasing total cholesterol, decreasingtriglyceride, reducing blood glucose level, enhancing HDL-Cholesterollevel, increasing the HDL-Cholesterol to total cholesterol ratio,lowering LDL-Cholesterol level, reducing the CRP level, decreasing theintima media thickening, reducing atherogenic index of plasma,increasing Apo A-1 levels, decreasing Apo B levels, decreasing Apo B toApo A-1 ratio, reducing Homocysteine level, reduction in glycosylatedHb, modulating TSH level, decreasing HMGCoA reductase activity, loweringtotal cholesterol without changing CoQ10 levels even at a lower dosagelevel.

TABLE B Amla seed blend composition of sample1 and product 1 indifferent ratios Sample 1 Product 1 Ratios Triterpenoids Hydroxycinnamicacid Fatty acids Sample 1:Product 1 (%) (%) (%) 1:5 10.6 4.5 64.6 1:221.2 9 51.6 2:3 25.4 10.8 46.2 1:1(Sample 2) 31.8 13.5 38.8 3:2 38.116.2 31 2:1 42.3 18 25.8 3:1 47.6 20.3 19.4 5:1 52.9 22.5 12.9 10:1 57.7 24.5 7.1 20:1  60.5 25.7 3.7

Some embodiments provide a dosage form of the first extract of seeds ofEmblica officinalis and a filler. The filler can be lactose, spray driedlactose, starch, dibasic calcium phosphate, tribasic calcium phosphate,microcrystalline cellulose, hydroxy propyl methyl cellulose, or calciumcarbonate, or combinations thereof. The dosage form can be capsule,tablet, granule, sachet, powder, paste, ointment, infusion, injection,ampoule, solution, suspension, emulsion, pills, oil, or, cream.

Some embodiments provide a dosage form of the first extract of seeds ofEmblica officinalis and a filler. In some embodiments, first extract ofseeds of Emblica officinalis and filler are blended in a ratio of firstextract of seeds of Emblica officinalis to filler ranging from about10:1 to about 3:1. In some embodiments, first extract of seeds ofEmblica officinalis and filler are blended in a ratio of first extractof seeds of Emblica officinalis to filler of about 5:1. In someembodiments, first extract of seeds of Emblica officinalis and fillerare blended in a ratio of first extract of seeds of Emblica officinalisto filler of about 6:1. In some embodiments, first extract of seeds ofEmblica officinalis and filler are blended in a ratio of first extractof seeds of Emblica officinalis to filler of about 4.5:1.

In another embodiment, 500 mg capsule having a first extract of seeds ofEmblica officinalis and filler includes a first extract to filler ratioof about 10:1 to about 3:1. Some embodiments have a ratio of firstextract to fillers of about 4.5:1. A 500 mg capsule of first extract ofseeds of Emblica officinalis with filler has about 400 mg to about 440mg of first extract of seeds of Emblica officinalis. Some embodimentshave about 430 mg of the first extract. Some embodiments have about 410mg of the first extract of seeds of Emblica officinalis. A 500 mgcapsule of first extract of seeds of Emblica officinalis with filler canhave about 60 mg to about 100 mg of filler. Some embodiments have about90 mg filler. A 500 mg capsule of first extract of seeds of Emblicaofficinalis and filler can have triterpenoids ranging from about 200 mgto about 240 mg and hydroxycinnamic acid ranging from about 80 mg toabout 100 mg. In some embodiments of the dosage form, the triterpenoidscan be about 213.5 mg and hydroxycinnamic acid can be about 90.8 mg.

Some embodiments provide a dosage form of the blend of extracts of seedsof Emblica officinalis and a filler. The blend of extracts of seeds ofEmblica officinalis has a first extract of seeds of Emblica officinalis,and, a second extract of Emblica Officinalis (referred to as Product 1from Example 13). The fillers include lactose, spray dried lactose,starch, dibasic calcium phosphate, tribasic calcium phosphate,microcrystalline cellulose, hydroxy propyl methyl cellulose, or calciumcarbonate, or combinations thereof. The dosage forms include capsule,tablet, granule, sachet, powder, paste, ointment, infusion, injection,ampoule, solution, suspension, emulsion, pills, oil, or, cream.

Some embodiments provide a dosage form of the blend of extracts of seedsof Emblica officinalis and a filler. The blend of extracts of seeds ofEmblica officinalis has a first extract of seeds of Emblica officinalis,and, a second extract of Emblica Officinalis (referred to as Product 1from Example 13). In some embodiments, blend of extracts of seeds ofEmblica officinalis and filler are blended in a ratio of blend ofextracts of seeds of Emblica officinalis to filler ranging from about10:1 to about 3:1. In some embodiments, blend of extracts of seeds ofEmblica officinalis and filler are blended in a ratio of blend ofextracts of seeds of Emblica officinalis to filler of about 5:1. In someembodiments, blend of extract of seeds of Emblica officinalis and fillerare blended in a ratio of blend of seeds of Emblica officinalis tofiller of about 6:1. In some embodiments, blend of extract of seeds ofEmblica officinalis and filler are blended in a ratio of blend of seedsof Emblica officinalis to filler of about 4.5:1.

In another embodiment, 500 mg capsule having blend of extract of seedsof Emblica officinalis and filler includes blend of extract of seeds ofEmblica officinalis to filler ratio of about 10:1 to about 3:1. Someembodiments have a ratio of blend of extract of seeds of Emblicaofficinalis to fillers of about 4.5:1. A 500 mg capsule of blend ofextract of seeds of Emblica officinalis with filler has about 400 mg toabout 440 mg of blend of extract of seeds of Emblica officinalis. Someembodiments have about 430 mg of the blend of extract of seeds ofEmblica officinalis. Some embodiments have about 410 mg of the blend ofextract of seeds of Emblica officinalis. A 500 mg capsule of blend ofextract of seeds of Emblica officinalis with filler can have about 60 mgto about 100 mg of filler. Some embodiments have about 90 mg filler. A500 mg capsule of blend of extract of seeds of Emblica officinalis andfiller can have triterpenoids ranging from about 100 mg to about 120 mg,hydroxycinnamic acid ranging from about 40 mg to about 55 mg and fattyacid ranging from about 120 mg to about 140 mg. In some embodiments ofthe dosage form, the triterpenoids can be about 107.6 mg,hydroxycinnamic acid can be about 45.4 mg and fatty acids can be about129.8 mg.

Some embodiments provide a method of preparing a first extract(sample 1) of seeds of Emblica officinalis. The method includesdeseeding fresh fruits of Emblica officinalis to obtain fresh seeds.Then crushing fresh seeds to obtain crushed seeds. Next, the crushedseeds are extracted with methanol to obtain a residue and a supernatant.The supernatant is concentrated to obtain a concentrated methanolextract. The concentrated methanol extract is dried to obtain a firstpowder. The first powder of methanol extract of seeds of Emblicaofficinalis is macerated with water to obtain a mixture. The mixture isextracted with ethyl acetate to obtain an ethyl acetate phase and anaqueous phase. The ethyl acetate phase is concentrated to obtain aconcentrated ethyl acetate extract. The concentrated ethyl acetateextract is dried to obtain a second powder. The second powder isrefluxed with 50% methanol to obtain a 50% methanol part and an oilpart. The 50% methanol part is concentrated to obtain a concentratedmethanol part. The concentrated methanol part is loaded onto an HP20column to obtain an extract loaded HP20 column. The loaded HP20 columnis eluted first with ethyl acetate and then eluted with 100% methanol toobtain an ethyl acetate elute and a methanol elute. The methanol eluteis concentrated to obtain a concentrated methanol elute. The methanolelute is dried to obtain a third powder, which is referred to as thefirst extract of seeds of Emblica officinalis. The first extract ofseeds of Emblica officinalis includes about 20% to about 70%triterpenoids, and, about 10% to about 40% hydroxycinnamic acids.

Some embodiments provide a method of preparing a blend of extracts ofseeds of Emblica officinalis. The blend includes the first extract ofseeds of Emblica officinalis and a second extract (some embodiments arereferred to as product 1) of seeds of Emblica officinalis. The method ofpreparing the blend includes blending the first extract of seeds ofEmblica officinalis and the second extract of seeds of Emblicaofficinalis in a weight ratio ranging from about 1:60 to about 99:1. Thesecond extract of seeds of Emblica officinalis is prepared by a methodincluding deseeding fresh fruits of Emblica officinalis to obtain seedsof Emblica officinalis. Next the seeds of Emblica officinalis arecrushed to obtain crushed seeds. The crushed seeds are extracted with95% methanol to obtain a residue and a supernatant. The supernatant isconcentrated to obtain a concentrated methanol extract. The concentratedmethanol extract is dried to obtain a powder of methanol extract ofseeds of Emblica officinalis. The powder of methanol extract of seeds ofEmblica officinalis is dispersed in water to obtain a dispersion. Thedispersion is extracted with hexane to obtain a water phase and a hexanephase. The hexane phase is concentrated to obtain a liquid form of aconcentrated hexane extract. The liquid form of the concentrated hexaneextract is cooled to obtain precipitates or crystals, and, a liquidportion. The liquid portion is separated from the precipitates orcrystals to obtain a liquid. This liquid is the second extract of seedsof Emblica officinalis. The second extract of seeds of Emblicaofficinalis includes alpha linolenic acid, linoleic acid and oleic acid.Method of preparing a first extract of seeds of Emblica officinalis(also referred to as sample 1), second extract of seeds of Emblicaofficinalis (also referred to as product 1) and a blend of the firstextract and the second extract of seeds of Emblica officinalis isprovided in FIGS. 14A and 14B.

Some embodiments provide a method of lowering total cholesterol byadministering the first extract of seeds of Emblica officinalis. Someembodiments provide a method of lowering triglycerides by administeringthe first extract of seeds of Emblica officinalis. Some embodimentsprovide a method of lowering VLDL cholesterol by administering the firstextract of seeds of Emblica officinalis. Some embodiments provide amethod of lowering LDL cholesterol by administering the first extract ofseeds of Emblica officinalis. Some embodiments provide a method ofenhancing HDL cholesterol by administering the first extract of seeds ofEmblica officinalis. Some embodiments provide a method of increasing aratio between HDL to total cholesterol by administering the firstextract of seeds of Emblica officinalis. Some embodiments provide amethod of reducing blood glucose level by administering an amla seedblend composition. Some embodiments provide a method of reducing hairfall by administering an amla seed blend composition. Some embodimentsprovide a method of decreasing atherogenic index of plasma byadministering the first extract of seeds of Emblica officinalis. Someembodiments provide a method of decreasing risk for coronary heartdisease by administering the first extract of seeds of Emblicaofficinalis. Some embodiments provide a method of decreasing atherogenicdyslipidemia by administering the first extract of seeds of Emblicaofficinalis. Some embodiments provide a method of decreasing the ratioof Apo B to Apo A1 by administering the first extract of seeds ofEmblica officinalis. Some embodiments provide a method of loweringhomocysteine level by administering the first extract of seeds ofEmblica officinalis. Some embodiments provide a method of loweringglycosylated hemoglobin level by administering the first extract ofseeds of Emblica officinalis. Some embodiments provide a method ofmodulating TSH levels by administering the first extract of seeds ofEmblica officinalis. Modulating refers to correcting the abnormal TSHlevels to normal range of TSH. For example, if a subject has a lowerthan normal TSH level, administering the extract would increase TSH butonly upto normal range and not to hyperthyroid range. However, if asubject has higher than normal TSH level, extract administration willlower the TSH to normal range but not to the hypothyroid range. In someembodiments, extract administration increases TSH levels. In someembodiments, it decreases TSH levels. And in some embodiments, TSH levelis not affected by extract administration. Some embodiments provide amethod of lowering HMGCoA reductase by administering the first extractof seeds of Emblica officinalis. In some embodiments a method oflowering total cholesterol without altering CoQ10 level by administeringthe first extract of seeds of Emblica officinalis is provided.

Some embodiments provide a method of lowering total cholesterol byadministering a blend of extracts of seeds of Emblica officinalis. Theblend includes the first extract of Emblica officinalis and the secondextract of Emblica officinalis. A method of lowering triglycerides byadministering the blend of extracts of seeds of Emblica officinalis isprovided. A method of lowering VLDL cholesterol administering the blendof extracts of seeds of Emblica officinalis. Some embodiments provide amethod of lowering LDL cholesterol by administering the blend ofextracts of seeds of Emblica officinalis. Some embodiments provide amethod enhancing HDL cholesterol by administering the blend of extractsof seeds of Emblica officinalis. Methods of increasing a ratio betweenHDL to total cholesterol by administering the blend of extracts of seedsof Emblica officinalis is provided. Some embodiments provide a method ofreducing blood glucose level by administering an amla seed blendcomposition. Some embodiments provide a method of reducing hair fall byadministering an amla seed blend composition. Methods of decreasingatherogenic index of plasma by administering the blend of extracts ofseeds of Emblica officinalis is provided. Methods of decreasing risk forcoronary heart disease by administering the blend of extracts of seedsof Emblica officinalis is provided. Methods of decreasing atherogenicdyslipidemia by administering the blend of extracts of seeds of Emblicaofficinalis is provided. Method of decreasing ratio of Apo B to Apo A1by administering the blend of extracts of seeds of Emblica officinalisis provided. Methods of lowering homocysteine level by administering theblend of extracts of seeds of Emblica officinalis is provided. Methodsof lowering glycosylated hemoglobin level by administering the blend ofextracts of seeds of Emblica officinalis is provided. Methods ofmodulating TSH by administering the blend of extracts of seeds ofEmblica officinalis is provided. Methods of lowering HMGCoA reductase byadministering the blend of extracts of seeds of Emblica officinalis isprovided. Methods for lowering total cholesterol without altering CoQ10level by administering the blend of extracts seeds of Emblicaofficinalis is provided.

It will be readily understood by the skilled artisan that numerousalterations may be made to the examples and instructions given herein.These and other objects and features of the invention will be madeapparent from the following examples. The following examples asdescribed are not intended to be construed as limiting the scope of thepresent invention.

Example 1 Method of Preparation of Ethyl Acetate Extract of MethanolExtract of Seed of Emblica officinalis

500 Kg of fresh fruits of Emblica officinalis (Amla) were collected.Fruits were deseeded by deseeding machine and 75 Kg of fresh seedsobtained were crushed through roller mill. 95% Methanol in an amount 2times the quantity of crushed seeds was added to the crushed seeds toform a mixture for methanol extraction. The extraction was performedusing an extractor with reflux condenser. The bottom of the extractorwas fitted with a polypropylene (100 microns) filter cloth. The mixturewas refluxed for one hour at 65° C. to obtain a first residue andsupernatant. The residue and supernatants were separated by draining outthe supernatant from the extractor bottom through the polypropylenefilter cloth using a centrifugal pump. After the first extraction, thefirst residue was further extracted with two times the quantity ofmethanol at 65° C. to get second residue and supernatant. The secondresidue was further extracted with two times the quantity of methanol at65° C. to get third residue and supernatant. All the supernatants werepooled and concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 65° C. to form concentrated methanol extract.Concentrated methanol extract was dried under vacuum at above 500 mm ofmercury to obtain 5 kg of powder of methanol extract of seed of Emblicaofficinalis.

The powder of methanol extract of seed of Emblica officinalis wasmacerated with water and transferred into a liquid-liquid extractor andextracted with ethyl acetate. Ethyl acetate phase and aqueous phase wereseparated. After extraction ethyl acetate phase was collected. Ethylacetate phase was concentrated in an Agitated thin film evaporator toform concentrated ethyl acetate extract. Ethyl acetate concentrate wasfed into vacuum stripper and dried under vacuum at above 500 mm ofmercury to obtain 2.5 kg of powder of ethyl acetate extract of methanolextract of seed of Emblica Officinalis. Method of preparing the extractis provided in FIG. 1.

Powder of ethyl acetate extract of methanol extract of seed of Emblicaofficinalis had 9.5% triterpenoids, 4.3% hydroxycinnamic acids and 41.8%fatty acids.

2 Kg powder of ethyl acetate extract of methanol extract of seed ofEmblica officinalis was blended with 0.45 kg of microcrystallinecellulose in a Double Cone Blender (stainless steel SS-316, capacity2000 litre, manufacturer: Zebra Pharma, Mumbai) for 1 hour to obtain atotal of 2.45 kg blend of powder of ethyl acetate extract of methanolextract of seed of Emblica officinalis and microcrystalline cellulose.

2.45 Kg blend composition had 7.8% triterpenoids, 3.5% hydroxycinnamicacids and 34.3% fatty acids.

Example 2 Method of Preparation of Ethyl Acetate Extract of Seed ofEmblica officinalis

500 Kg of fresh fruits of Emblica officinalis were collected. Fruitswere deseeded by deseeding machine and 75 Kg fresh seeds obtained werecrushed through roller mill. Crushed seeds were filled in the Soxhletextractor and extracted with ethyl acetate (300 L). The extraction wascarried out for 5 hrs at a temperature of about 78° C. After thecompletion of extraction, the supernatant was filtered and concentratedin an Agitated thin film evaporator (ATFE) at a temperature of 75° C. toform concentrated ethyl acetate extract. Concentrated ethyl acetateextract was dried under vacuum at above 500 mm of mercury to get 2 Kg ofpowder of ethyl acetate extract of seed of Emblica officinalis. Methodof preparing the Emblica officinalis extract is provided in FIG. 2.

Example 3 Method of Preparation of Pectinase Treated Water Extract ofFruits of Emblica officinalis

100 Kg of fresh fruits of Emblica officinalis were collected. Freshfruits of Emblica officinalis were pulped with demineralized water tocreate slurry. The slurry was treated with pectinase and then filteredto obtain a solution. The solution was concentrated and dried undervacuum. 5 Kg of dried product was pulverized and sieved through 30meshes to obtain a powder of a pectinase treated water extract ofEmblica officinalis.

Example 4 Method of Preparation of Alcoholic Extract of Fruits ofEmblica officinalis

100 Kg of fresh fruits of Emblica officinalis were collected. Freshfruits of Emblica officinalis were pulverized. 95% Methanol in an amount2 times the quantity (200 L) of pulverized fruits were added to form amixture for methanol extraction. The extraction was performed using anextractor with reflux condenser. The bottom of the extractor was fittedwith a polypropylene (100 microns) filter cloth. The mixture wasrefluxed for one hour at 65° C. to obtain a residue and supernatant. Theresidue and supernatants were separated by draining out the supernatantfrom the extractor bottom through the polypropylene filter cloth using acentrifugal pump. After the first extraction, the residue was furtherextracted with two times the quantity of methanol at 65° C. to get aresidue and a supernatant. The residue was further extracted with twotimes the quantity of methanol at 65° C. to get third residue andsupernatant. All the supernatants were pooled and concentrated in anAgitated thin film evaporator (ATFE) at a temperature of 65° C. to formconcentrated methanol extract. Concentrated methanol extract was driedunder vacuum at above 500 mm of mercury to obtain 5 kilograms of powderof methanol extract of fruit of Emblica Officinalis. Method of preparingextract is provided in FIG. 4.

Example 5 Method of Preparation of Pectinase Treated Water Extract ofDeseeded Emblica officinalis

100 Kg of fresh fruits of Emblica officinalis were collected. Freshfruits of Emblica officinalis were deseeded by deseeding machine toobtain 85 Kg deseeded fruit. Deseeded Emblica officinalis fruits werepulped with demineralized water to create slurry. The slurry was treatedwith pectinase and then filtered to obtain a solution. The solution wasconcentrated and dried under vacuum. 4 Kg of dried product waspulverized and sieved through 30 meshes to obtain a powder of apectinase treated water extract of deseeded Emblica officinalis. Methodof preparing extract is provided in FIG. 5.

Example 6 Method of Preparation of Alcoholic Extract of Deseeded Emblicaofficinalis

100 Kg of fresh fruits of Emblica officinalis were collected. Freshfruits of Emblica officinalis were deseeded by deseeding machine toobtain 85 Kg deseeded Emblica officinalis fruits. Deseeded Emblicaofficinalis fruits were pulverized. 95% methanol in an amount 2 timesthe quantity (170 L) of pulverized fruits was added to form a mixturefor methanol extraction. The extraction was performed using an extractorwith reflux condenser. The bottom of the extractor was fitted with apolypropylene (100 microns) filter cloth. The mixture was refluxed forone hour at 65° C. to obtain a first residue and supernatant. Theresidue and supernatants were separated by draining out the supernatantfrom the extractor bottom through the polypropylene filter cloth using acentrifugal pump. After the first extraction, the first residue wasfurther extracted with two times the quantity of methanol at 65° C. toget second residue and supernatant. The second residue was furtherextracted with two times the quantity of methanol at 65° C. to get thirdresidue and supernatant. All the supernatants were pooled andconcentrated in an Agitated thin film evaporator (ATFE) at a temperatureof 65° C. to form concentrated methanol extract. Concentrated methanolextract was dried under vacuum at above 500 mm of mercury to obtain 3.5kilograms of powder of methanol extract of deseeded fruit of Emblicaofficinalis. Method of preparing extract is provided in FIG. 6.

Example 7 Method of Preparation of Powder of Dried Amla Seed

10 Kg of fresh fruits of Emblica officinalis (Amla) were collected.Fruits were deseeded by deseeding machine and 1.5 Kg of fresh seedsobtained was dried in tray drier at 40° C. The dried material waspowdered to obtain 0.75 Kg powder of dried seed of Emblica officinalis.Method of preparing extract is provided in FIG. 7.

Example 8 Method of Preparation of Water Extract of Dried Amla Seed

500 Kg of fresh fruits of Emblica officinalis (Amla) were collected.Fruits were deseeded by deseeding machine to obtain 75 Kg fresh seeds.Fresh seeds were dried in tray drier at 40° C. The dried seeds werecrushed and charged in to an extractor. Around 200 Litres of water wasadded into the crushed seed and kept for a contact time of 3 hrs. Thenthe water part was collected and fresh water was again added into seedsand repeated the extraction thrice. All the water parts were pooled,filtered and concentrated in an evaporator, when the concentrated waterextract of dried seed reached the bottom of the vessel, the concentratewas fed into drier and dried under vacuum above 500 mm of mercury. Driedproduct was discharged from the bottom of the vessel and pulverized toobtain 4 Kg powder of the water extract of dried seed of Emblicaofficinalis. Method of preparing extract is provided in FIG. 8.

Example 9 Method of Preparation of Methanol Extract of Dried Amla Seed

500 Kg of fresh fruits of Emblica officinalis (Amla) were collected.Fruits were deseeded by deseeding machine to obtain 75 Kg fresh seeds.Fresh seeds were dried in tray drier at 40° C. The dried seeds werecrushed and charged into an extractor. Around 200 litres of 95% methylalcohol was pumped into the extractor and kept for a contact time of 3hours. Then the solvent part (methanol part) was collected and freshmethyl alcohol pumped again into the extractor and extraction repeatedthrice. All the extracts (methanol part) were pooled, filtered and driedin an Agitated thin film drier (ATFD) which was working under vacuum 700mm Mercury. Dried product was discharged from the bottom of the vesseland then pulverized to obtain 5 Kg powder of an alcoholic extract ofseed of Emblica officinalis. The method of preparing extract is providedin FIG. 9.

Example 10 Method of Preparation of Powder of Dried Amla Fruit

Fresh fruits of Emblica officinalis 100 kgs were washed and chopped intoflakes and dried in a hot air oven at around 110° C. for 10 hours. 9 Kgof dried material was powdered to obtain a powder of dried fruits ofEmblica officinalis. The method of preparing extract is provided in FIG.10.

Example 11 Method of Preparation of Water Extract of Dried Amla Fruit

100 Kg of fresh fruits of Emblica officinalis were collected. Freshfruits were washed and chopped into flakes and dried in a hot air ovenat around 110° C. for 10 hours. Dried flakes were charged in to anextractor and around 200 Liters of water was added into the dried flakesand kept for a contact time of 3 hrs. Then the water part was collectedand water was again added into flakes and repeated thrice. All the waterparts were pooled, filtered and concentrated in an evaporator, when theconcentrated water extract of dried fruit reached the bottom of thevessel, the concentrate was fed into drier and dried under vacuum above500 mm of mercury. 6 Kg of dried product was discharged from the bottomof the vessel and pulverized to obtain a powder of the water extract ofdried fruits of Emblica officinalis. The method of preparing extract isprovided in FIG. 11.

Example 12 Method of Preparation of Methanol Extract of Dried Amla Fruit

100 Kg of fresh fruits of Emblica officinalis were collected. Freshfruits were washed and chopped into flakes and dried in a hot air ovenat around 110° C. for 10 hours. Dried flakes were charged in to anextractor and around 200 liters of 95% methyl alcohol was pumped intothe extractor and kept for a contact time of 3 hours. Then the solventpart (methanol part) was collected and fresh methyl alcohol pumped againinto the extractor and extraction repeated thrice. All the extracts(methanol part) were pooled, filtered and dried in an Agitated thin filmdrier (ATFD) which was working under vacuum 700 mm Mercury. 5 Kg ofdried product was discharged from the bottom of the vessel and thenpulverized to obtain of powder of an alcoholic extract of fruits ofEmblica officinalis. The method of preparing extract is provided in FIG.12.

Example 13 Method of Enrichment of Triterpinoids and Hydroxycinimic AcidBlending with Fatty Acid in a Amla Seed Extract

500 Kg of fresh fruits of Emblica officinalis (Amla) were collected.Fruits were deseeded by deseeding machine to obtain 75 Kg of fresh seedsof amla. The fresh seeds were crushed through roller mill. 95% methanolin an amount 2 times the quantity of crushed seeds was added to thecrushed seeds to form a mixture for methanol extraction. The extractionwas performed using an extractor with reflux condenser. The bottom ofthe extractor was fitted with a polypropylene (100 microns) filtercloth. The mixture was refluxed for one hour at 65° C. to obtain a firstresidue and supernatant. The first residue and supernatant wereseparated by draining out the supernatant from the extractor bottomthrough the polypropylene filter cloth using a centrifugal pump. Afterthe first extraction, the first residue was further extracted with twotimes the quantity of methanol at 65° C. to get a second residue andsupernatant. The second residue was further extracted with two times thequantity of methanol at 65° C. to get a third residue and supernatant.All the supernatants were pooled and concentrated in an Agitated thinfilm evaporator (ATFE) at a temperature of 65° C. to form a concentratedmethanol extract. The concentrated methanol extract was dried undervacuum at above 500 mm of mercury to obtain 5 kg of powder of methanolextract of seed of Emblica officinalis.

The powder of methanol extract of seed of Emblica officinalis wasdispersed in water and transferred into a liquid-liquid extractor andextracted with hexane. After extraction hexane phase and aqueous phaseseparated. Then the hexane phase was collected through side valve.Hexane phase was concentrated in an Agitated thin film evaporator toform 2 Kg of concentrated hexane extract. The concentrated hexaneextract was cooled at 4° C. and kept cold for 24 hrs. Some components ofthe concentrated hexane extract precipitated or crystallized by thiscooling. The crystals or precipitates were separated from the liquidcooled concentrated hexane extract by passing the cooled concentratedhexane extract through a filter press. The precipitates or crystals werefound to contain high melting point components such as palmitic acid andstearic acid. 1.5 kilograms of liquid (product 1) was obtained afterpassing through the cooled concentrated hexane extract in the filterpress. Product 1 was found to contain 77.5% unsaturated fatty acids suchas 34.2% alpha-linolenic acid, 26.3% linolenic acid, and 16.5% oleicacid.

Ethyl acetate was added to aqueous phase in a liquid-liquid extractorfor extraction. After extraction ethyl acetate phase and aqueous phasewere separated and ethyl acetate phase was collected through side valve.Ethyl acetate phase was concentrated in an Agitated thin film evaporatorto form concentrated ethyl acetate extract. Ethyl acetate concentratewas fed into vacuum stripper and dried under vacuum at above 500 mm ofmercury to obtain 2.5 kilograms of powder of ethyl acetate extract ofmethanol extract of seed of Emblica officinalis. This extract was foundto contain triterpenoids and polyphenols. Polyphenols includedhydroxycinnamic acid.

Powder of ethyl acetate extract of methanol extract of seed of Emblicaofficinalis was mixed with water and loaded in a column having a FPX 66ion-exchange resin (Rohm & Haas, Philadelphia, USA). The columnchromatography was performed to further purify the triterpenoids, and,to separate hydroxycinnamic acids from other polyphenols. Column wasinitially eluted with water and water fraction was collected. Then thecolumn was eluted with different concentrations of methanol (50%methanol and 80% methanol) and collected the different methanolfractions. Water fraction was concentrated and dried under vacuum toform of powder of water elute of ethyl acetate extract of seed ofEmblica officinalis with an yield of 0.1 Kg denoted as Fraction 1.Fraction 1 was found to contain small amount of triterpenoids which wasconfirmed by HPLC. The 50% methanol fraction was concentrated and driedunder vacuum to form powder of 50% methanol elute of ethyl acetateextract of seed of Emblica officinalis with an yield of 1 kg denoted asFraction 2. Fraction 2 was found to contain triterpenoids and it wasconfirmed by HPLC.

80% methanol fraction was concentrated and dried under vacuum to formpowder of 80% methanol elute of ethyl acetate extract of seed of Emblicaofficinalis with an yield of 0.75 kg denoted as Fraction 3. Fraction 3was found to contain hydroxycinnamic acids confirmed by HPLC method.

Fractions 1, 2 and 3 were combined to form a product 2. Product 2 wasblended with product 1 in a 2:3 ratio to form an amla seed blend product3 with an yield of 3.35 Kg. The method of preparing extract is providedin FIG. 13. Product 3 had about 20% triterpenoids, about 11.7%hydroxycinnamic acids, about 46.2% unsaturated fatty acids and about0.3% saturated fatty acids. Product 3 included about 9% beta sitosterol, about 6% beta amyrin, about 5% lupeol, about 7.5% ferulic acid,about 4.2% coumaric acid, about 20.5% alpha linolenic acid, about 15.8%linolenic acid, about 9.9% oleic acid, about 0.2% stearic acid and about0.1% palmitic acid.

Example 14 Analysis of Triterpenoids by HPLC

The triterpenoids was estimated by high performance liquidchromatography (HPLC-DAD) on a C18 column (250×4.6 mm). The mobile phasewas acetonitrile used under isocratic condition with an eluent flow rateof 1 ml/min.

Standard was prepared by weighing 5 mg of standards lupeol, beta amyrinand betasitosterol (95% purity) and was made up to 10 ml withacetonitrile and stored in darkness at 4° C. Sample was prepared byweighing 50 mg of the extract and was made up to 50 ml with acetonitrileand stored in darkness at 4° C. Both the sample and standard werefiltered separately through a 0.2 μm membrane filter before injectioninto the HPLC column. The injection volume was 20 μl. The triterpenoidswas detected at 210 nm. By comparing the area of standard and sample,the percentage of triterpenoids present in the sample was quantified.(Separation and identification of some common isomeric planttriterpenoids by thin-layer chromatography and high-performance liquidchromatography, Mitja et al, J Chromatogr A 2009 Sep. 18;1216(38):6662-70. doi: 0.1016/j.chroma.2009.07.038. Epub 2009 Jul. 29.).

Example 15 Analysis of Hydroxycinnamic Acids by HPLC

The hydroxycinnamic acids were estimated by high performance liquidchromatography (HPLC-DAD) on a C18 column (250×4.6 mm). The mobile phasewas Solvent A-1% acetic acid in water, solvent B-1% aceticacid/water/acetonitrile (2:68:30). Gradient: 0 min. 7% B increased to90% B. Flow rate 1 ml/min. Detection at 320 nm.

Standard was prepared by weighing 5 mg of standards ferulic acid andp-coumaric acid (95% purity) and was made up to 10 ml with methanol andstored in darkness at 4° C. Sample was prepared by weighing 50 mg ofextract and was made up to 50 ml with methanol and stored in darkness at4° C. Both the sample and standard were filtered separately through a0.2 μm membrane filter before injection into the HPLC column. Theinjection volume was 20 μl. The hydroxycinnamic acids were detected at320 nm. By comparing the area of standard and sample, the percentage ofhydroxycinnamic acids present in the sample was quantified. (A “NovelProtocol for the Analysis of Hydroxycinnamic Acids in Leaf Tissue ofChicory (Cichorium intybus L., Asteraceae),http://www.hindawi.com/45870319/Meriem Bahri et al, the scientific worldjournal, volume 2012, Article ID 142983.)

Example 16 Analysis of Fatty Acids by GC

Fatty acids were analysed by gas chromatography method. 250 mg ofstandard oil fatty acid methyl ester was weighed in 25 ml standard flaskand made up to 25 ml with isooctane. 1 microlitre of the standardsolution was injected in GC. Retention time of each component was foundout.

0.3 gm of the sample (extract) was weighed into a 100 ml RB flask and1.5 ml of 0.5 N methanolic NaOH was added. Sample was kept in a boilingwater bath with a water condenser and heated at 100° C. for 5 minute.Cooled and 2 ml of boron tri fluoride (BF3)-Methanol solution was addedand heated at 100° C. for 30 minutes. Cooled to 30-40° C. and 5 mlisooctane was added and shook vigorously for 30 seconds.

5 ml of saturated NaCl solution was added immediately and shookvigorously and cooled to room temperature. Iso-octane layer wasseparated and aqueous layer was again extracted with iso-octane.Isooctane layers were mixed and dried. Then made up the residue to 25 mlwith iso-octane and 2 micro litre was injected in GC. Retention time ofeach component was found out and compared with components of standardwith the same retention time. By comparing the area of standard andsample, the percentage of fatty acids present in the sample wasquantified. (European Pharmacopoeia, fifth edition, vol 1, ISBN:92-871-5281-0, p-110).

Example 17 Determination of Polyphenol Content

The polyphenol was estimated by high performance liquid chromatography(HPLC-DAD) on a C18 column (250×4.6 mm). The mobile phase was SolventA-0.1% Trifluro acetic acid in water, solvent B-Methanol. Isocratic(90:10) Flow rate 1 ml/min. Detection at 254 nm.

Standard was prepared by weighing 5 mg of standards gallic acid andellagic acid (95% purity) and was made up to 10 ml with methanol andstored in darkness at 4° C. Sample was prepared by weighing 50 mg of theextract and was made up to 50 ml with methanol and stored in darkness at4° C. Both the sample and standard were filtered separately through a0.2 μm membrane filter before injection into the HPLC column. Theinjection volume was 20 μl. The polyphenol was detected at 254 nm. Bycomparing the area of standard and sample, the percentage of polyphenolpresent in the sample was quantified. (HPLC Profiles of StandardPhenolic Compounds Present in Medicinal Plants, Gupta et al,International Journal of Pharmacognosy and Phytochemical Research 2012;4(3); 162-167.)

Example 18 Screening of Hypolipidemic Activity of Amla Seed ExtractUsing Triton WR 1339 Induced Dyslipidemia Model

Twenty four male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intofour groups Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only).

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal).

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (10 mg/kg, per oral).

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin (10 mg/kg, per oral).

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 1(A) Lipid profile (mg/dl) of rats treated with Amla seed extract.TC TG HDL Groups Before After Before After Before After Normal control.111.87 134.77 52.20 62.03 37.80 58.03 Triton only. 108.3 265.6 43.06692.7 36.13 49.5 Triton + Amla 117.2 120.87 58.63 152.23 39.4 47.27 seedextract (10 mg/Kg). Triton + 100.4 131.9 44.26 109.67 29.13 32.37Atorvastatin (10 mg/kg).

TABLE 1(B) Lipid profile (mg/dl) of rats treated with Amla seed extract.LDL VLDL Groups Before After Before After Normal control. 63.69 64.3710.44 12.41 Triton only. 63.59 77.59 8.62 138.55 Triton + Amla seedextract 66.03 43.15 11.72 30.44 (10 mg/Kg). Triton + Atorvastatin 62.4377.61 8.85 21.93 (10 mg/kg).

Results indicated that before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Injecting Triton alone (Group II) significantly increasedthe total cholesterol level to 265.6 mg/dl. Whereas in Group III,simultaneous oral administration of amla seed extract in triton injectedrats, cholesterol level was 120.87 mg/dl which was 2.2 times lower ascompared to triton alone group.

In group II triton injection increased the triglyceride level to 692.7mg/dl. Whereas in Group III, simultaneous oral administration of amlaseed extract in triton injected rats, triglyceride level was 152.23mg/dl which was 4.5 times lower as compared to triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.18. Whereas in Group III, simultaneous oraladministration of amla seed extract in triton injected rats, the ratioof HDL cholesterol to total cholesterol was 0.39 which was 2.2 timeshigher as compared to triton alone group.

In group II triton injection increased the LDL level to 77.59 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract in triton injected rats, LDL level was 43.15 mg/dl which was 1.8times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 138.55 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract in triton injected rats, VLDL level was 30.44 mg/dl which was4.6 times lower as compared to triton alone group.

The standard drug Atorvastatin was also effective in lowering thecholesterol as well as triglyceride level. These results clearlyindicate the hypolipidemic activity of amla seed extract in tritoninduced hyperlipidemia in rats.

Example 19 Hypolipidemic Activity of Amla Seed Extracts in DifferentDoses Using Triton WR 1339 Induced Dyslipidemia Model

Sixty male Albino rats (Sprague Dawley strain) weighing approximate250-300 gm were selected for the study. The animals were kept in theanimal house maintained at temp 24±2° C., 65% relative humidity and 12hr light/dark cycle. The rats were acclimatized for two weeks and duringthis period they had access to standard pellet diet and water adlibitum. After two weeks of acclimatization, all the rats were fastedovernight before injecting Triton WR 1339 (Tyloxapol) and administrationof test extracts/standard. The animals were divided into ten groups.Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only).

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal).

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (10 mg/kg, per oral).

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (7.5 mg/kg, per oral).

Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (5 mg/kg, per oral).

Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (2.5 mg/kg, per oral).

Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (2 mg/kg, per oral).

Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (1 mg/kg, per oral).

Group IX: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepare as per example 1 (0.5 mg/kg, per oral).

Group X: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byatorvastatin (10 mg/kg, per oral).

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 2(A) Lipid profile (mg/dl) of rats treated with amla seed extractin different doses. TC TG HDL Groups Before After Before After BeforeAfter Normal control. 105.52 111.36 49.38 40.55 30.12 33.67 Triton only.110.53 233.76 50.5 504.3 46.3 54.66 Triton + amla seed extract 10 mg/Kg.111.1 108.23 51.23 91.1 45.46 48.66 Triton + amla seed extract 7.5mg/Kg. 91.9 114 62.53 171.18 30.41 36.51 Triton + amla seed extract 5mg/Kg. 85.46 112.26 45.61 120.43 29.68 40.33 Triton + amla seed extract2.5 mg/Kg. 104.66 116.25 55.28 116.31 36.41 43.28 Triton + amla seedextract 2 mg/Kg. 96.35 118.54 49.53 105.56 38.55 41.26 Triton + amlaseed extract 1 mg/Kg. 101.25 121.55 58.33 125.56 40.25 42.58 Triton +amla seed extract 0.5 mg/Kg. 110.66 126.61 55.48 122.28 46.26 47.41Triton + atorvastatin 10 mg/kg. 87.43 120.83 39.9 103.86 31.66 39.83

TABLE 2(B) Lipid profile (mg/dl) of rats treated with amla seed extractin different doses. LDL VLDL Groups Before After Before After Normalcontrol. 67.55 71.64 9.85 8.09 Triton only. 47.46 78.24 10.09 100.86Triton + amla seed extract 10 mg/Kg. 55.4 38.76 10.24 18.13 Triton +amla seed extract 49.03 43.25 12.50 34.06 7.5 mg/Kg. Triton + amla seedextract 5 mg/Kg. 46.68 47.85 9.12 24.08 Triton + amla seed extract 57.2349.73 11.05 23.26 2.5 mg/Kg. Triton + amla seed extract 2 mg/Kg. 47.956.17 9.90 21.11 Triton + amla seed extract 1 mg/Kg. 49.34 53.86 11.6625.11 Triton + amla seed extract 53.31 54.75 11.09 24.45 0.5 mg/Kg.Triton + atorvastatin 10 mg/kg. 47.75 60.23 7.98 20.77

Results indicated that intraperitoneal injection of triton significantlyincreased the total cholesterol level about 2 times over the baselinevalue. Similarly, triglyceride level also increased to very high level(Group II) following triton injection. Simultaneous oral administrationof various doses of amla seed extract in triton injected rats loweredthe cholesterol increase to almost normal level (Group III to IX).Triglyceride level was also lowered by all the doses of triton with amlaseed extract as compared to triton alone group. The standard drugatorvastatin was also effective in lowering the cholesterol as well astriglyceride level (Group X). These results clearly indicate thehypolipidemic activity of amla seed extract at various dose levels intriton induced hyperlipidemia in rats.

Example 20 Hypolipidemic Activity of Amla Seed Extract Compared withOther Amla Extracts Using Triton WR 1339 Induced Dyslipidemia Model

Forty eight male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intoeight groups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only).

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal).

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepared as per example 1 (2.5 mg/kg, per oral).

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 3 (40 mg/kg, per oral).

Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 4 (40 mg/kg, per oral).

Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 5 (40 mg/kg, per oral).

Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 6 (40 mg/kg, per oral).

Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byatorvastatin (10 mg/kg, per oral).

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 3(A) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts. TC TG HDL Groups Before After Before AfterBefore After Normal control. 95.52 99.36 42.38 48.51 31.02 32.11 Tritononly. 102.52 348.87 61.51 614.21 41.90 51.22 Triton + amla seed extractprepared 108.22 112.23 61.23 95.88 39.46 47.51 as per example 1 (2.5mg/Kg). Triton + amla extract prepared as 96.52 128.25 54.55 102.3444.25 48.35 per example 3 (40 mg/kg). Triton + amla extract prepared as108.24 124.55 59.32 115.36 41.26 46.52 per example 4 (40 mg/kg).Triton + amla extract prepared as 100.66 135.36 65.25 132.22 38.66 40.11per example 5 (40 mg/kg). Triton + amla extract prepared as 97.56 132.6660.21 125.54 34.59 36.54 per example 6 (40 mg/kg). Triton + atorvastatin(10 mg/kg). 97.33 132.58 48.99 218.66 35.11 33.84

TABLE 3(B) Lipid profile (mg/dl) of rats treated with amla seed extractand amla fruit extracts. LDL VLDL Groups Before After Before AfterNormal control. 56.03 57.54 8.47 9.70 Triton only. 48.32 174.80 12.30122.84 Triton + amla seed extract 56.52 45.55 12.24 19.17 prepared asper example 1 (2.5 mg/Kg). Triton + amla extract prepared as 41.36 59.4310.91 20.46 per example 3 (40 mg/kg). Triton + amla extract prepared as55.11 54.95 11.86 23.07 per example 4 (40 mg/kg). Triton + amla extractprepared as 48.95 68.80 13.05 26.44 per example 5 (40 mg/kg). Triton +amla extract prepared as 50.92 71.01 12.04 25.10 per example 6 (40mg/kg). Triton + atorvastatin (10 mg/kg). 52.43 55.00 9.79 43.73

In this study, effectiveness of amla seed extract (2.5 mg of extract/kgof subject) was compared with other amla extracts made as in Examples 3to 6.

Results indicated that before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Injecting Triton alone (Group II) significantly increasedthe total cholesterol level to 348.87 mg/dl. Whereas in Group III,simultaneous oral administration of amla seed extract in triton injectedrats, cholesterol level was 112.23 mg/dl which was 3.1 times lower ascompared to triton alone group.

In group II triton injection increased the triglyceride level to 614.21mg/dl. Whereas in Group III, simultaneous oral administration of amlaseed extract in triton injected rats, triglyceride level was 95.88 mg/dlwhich was 6.4 times lower as compared to triton alone group.

In triton alone group after triton injection, the ratio of HDL to totalcholesterol was 0.14. Whereas in Group III, simultaneous oraladministration of amla seed extract in triton injected rats, the ratioof HDL cholesterol to total cholesterol was 0.42 which was 3 timeshigher as compared to triton alone group.

In group II triton injection increased the LDL level to 174.8 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract in triton injected rats, LDL level was 45.55 mg/dl which was 3.8times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 122.84 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract in triton injected rats, VLDL level was 19.17 mg/dl which was6.4 times lower as compared to triton alone group.

The other amla extracts (prepared in example 3 to 6) at 40 mg/kg werealso able to decrease the cholesterol and triglyceride level but thesewere less effective than amla seed extract prepared in example 1. Thestandard drug Atorvastatin was also effective in lowering thecholesterol as well as triglyceride levels (Group VIII).

Example 21 Single Dosage Study for Evaluating Hypolipidemic Activity ofAmla Seed Extract Compared with Other Amla Extracts Using Triton WR 1339Induced Dyslipidemia Model

Forty eight male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intoeight groups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only).

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal).

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaseed extract prepared as per example 1 (2.5 mg/kg, per oral).

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 3 (2.5 mg/kg, per oral).

Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 4 (2.5 mg/kg, per oral).

Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 5 (2.5 mg/kg, per oral).

Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by amlaextract prepared as per example 6 (2.5 mg/kg, per oral).

Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byatorvastatin (10 mg/kg, per oral).

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 4(A) Lipid profile (mg/dl) of rats treated with amla seed extractand amla fruit extracts in same dosage level TC TG HDL Groups BeforeAfter Before After Before After Normal control. 102.25 101.56 56.3354.96 39.65 42.51 Triton only. 91.55 385.54 55.69 644.65 44.52 59.54Triton + amla seed extract prepared 94.66 108.25 60.23 111.36 38.4146.23 as per example 1 (2.5 mg/Kg). Triton + amla extract prepared as100.58 145.24 51.63 140.63 41.55 42.65 per example 3 (2.5 mg/kg).Triton + amla extract prepared as 106.52 139.66 49.36 131.33 42.36 46.31per example 4 (2.5 mg/kg). Triton + amla extract prepared as 95.66159.65 68.55 151.33 35.65 37.63 per example 5 (2.5 mg/kg). Triton + amlaextract prepared as 98.69 152.36 67.96 148.65 37.55 39.54 per example 6(2.5 mg/kg). Triton + atorvastatin (10 mg/kg). 99.83 122.54 46.59 211.0639.51 36.66

TABLE 4(B) Lipid profile (mg/dl) of rats treated with amla seed extractand amla fruit extracts in same dosage level. LDL VLDL Groups BeforeAfter Before After Normal control. 51.33 48.06 11.26 10.99 Triton only.35.89 197.07 11.13 128.93 Triton + amla seed extract 44.20 39.75 12.0422.27 prepared as per example 1 (2.5 mg/Kg). Triton + amla extractprepared as 48.70 74.47 10.32 28.12 per example 3 (2.5 mg/kg). Triton +amla extract prepared as 54.28 67.09 9.87 26.26 per example 4 (2.5mg/kg). Triton + amla extract prepared as 46.3 91.76 13.71 30.26 perexample 5 (2.5 mg/kg). Triton + amla extract prepared as 47.54 83.0913.59 29.73 per example 6 (2.5 mg/kg). Triton + atorvastatin (10 mg/kg).51.00 43.67 9.31 42.21

In this study, amla seed extract (2.5 mg extract/kg of subject) wascompared with other amla extracts made as in example 3 to 6 at the samedose level, i.e. 2.5 mg of extract/kg of subject.

Results indicated that before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Injecting Triton alone (Group II) significantly increasedthe total cholesterol level to 385.54 mg/dl. Whereas in Group III,simultaneous oral administration of amla seed extract in triton injectedrats, cholesterol level was 108.25 mg/dl which was 3.6 times lower ascompared to triton alone group.

In group II triton injection increased the triglyceride level to 644.65mg/dl. Whereas in Group III, simultaneous oral administration of amlaseed extract in triton injected rats, triglyceride level was 111.36mg/dl which was 5.8 times lower as compared to triton alone group.

In triton alone group after triton injection, the ratio of HDL to totalcholesterol was 0.15. Whereas in Group III, simultaneous oraladministration of amla seed extract in triton injected rats, the ratioof HDL cholesterol to total cholesterol was 0.42 which was 2.8 timeshigher as compared to triton alone group.

In group II triton injection increased the LDL level to 197.07 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract in triton injected rats, LDL level was 39.75 mg/dl which was 4.9times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 128.93 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract in triton injected rats, VLDL level was 22.27 mg/dl which was5.8 times lower as compared to triton alone group.

The other amla extracts (prepared in example 3 to 6) at 2.5 mg/kg wereable to decrease the cholesterol and triglyceride level to a certainextent only but these are not as effective as amla seed extract at samedose levels. The studies carried out indicate that the standard drugAtorvastatin was effective in lowering the cholesterol as well astriglyceride levels (Group VIII) but less effective when compared withthe amla seed extract.

Example 22 Antidiabetic Activity of Amla Seed Extract in StreptozotocinInduced Diabetic Rats

The amla seed extract as prepared in example 1 was evaluated forantidiabetic activity in experimental rats. Male/Female albino wistarrats were maintained as per standard guidelines: housed in polypropylenecages, under 12 hour artificial light and dark cycles at a temperatureof 24±2° C., given a standard pellet diet and water ad libitum. Theanimals were acclimatized to the animal house conditions for a weekbefore beginning the experiment.

Diabetes was induced by injecting streptozotocin 35 mg/kg dissolved in0.1M citrate buffer of pH 4.5, intra-peritoneally. Five days afterinduction of diabetes (day 1 of the study), animals were fasted for 12hours and the fasting blood glucose level (FBG) was estimated fordiagnosing diabetic rats. Animals with FBG above 200 mg/dl wereconsidered diabetic. The diabetic animals were randomly divided intothree groups of six animals each.

Following table 5 shows the treatment schedule given to the respectivegroup of animals for 28 days.

TABLE 5 Treatment schedule. Groups Drugs administered Group I Vehiclefor 28 days. Group II Glibenclamide (0.5 mg/kg) for 28 days. Group IIIAmla seed extract as per Example 1 (10 mg/kg) for 28 days.

Fasting blood glucose level and body weight of rats was measuredinitially and then at Day 7, Day 14, Day 21 and Day 28 of the study. Theplasma CRP level was measured initially and then at Day 28 of the study.

TABLE 6 Fasting blood glucose (FBG) level of diabetic rats treated withamla seed extract. Fasting Blood Glucose level (mg/dl) Day 1 Day Day DayDay Groups Treatment (Initial) 7 14 21 28 Group I Vehicle. 355 382 391411 431 Group II Glibenclamide. 406 211 179 136 109 Group III Amla seedextract 369 266 211 172 135 as per Example 1.

TABLE 7 CRP level of diabetic rats treated with amla seed extract. CRPlevel (mg/L) Day 1 Day 28 Groups Treatment (Initial) (Final) Group IVehicle. 10.3 11.3 Group II Glibenclamide. 10.8 9.6 Group III Amla seedextract as per Example 1. 9.9 6.3

As shown in the results, a single ip injection of streptozotocinincreased the blood glucose level to very high level and made the ratsdiabetic. Treatment of diabetic rats with amla seed extractsignificantly lowered the blood glucose level in 28 days. Thatcorresponds to a 2.7 times reduction in fasting blood glucose level indiabetic rats as compared to baseline (Day 1) after administration ofamla seed extract for 28 days. Treatment with glibenclamide alsodecreased the blood glucose level to nearly normal in 28 days. Bodyweight of diabetic rats treated with amla seed extract recoveredsignificantly as compared to vehicle group. The CRP level decreasedsignificantly in amla seed extract fed group but glibenclamide failed todecrease the CRP level significantly as compared to initial CRP value.28 days treatment with Amla seed extract to diabetic rats decreased theCRP level 1.6 times than initial level.

Example 23 Hypolipidemic Activity of Amla Seed Extract in CholesterolFed Rabbits

The amla seed extract as prepared in example 1 was evaluated forhypolipidemic activity in experimental rabbits. Male NZ white rabbitsweighing 1.5-2.0 kg were used for the experiment. They were housed in atemperature-controlled room (25±2° C.) in clean stainless steel cageswith ‘12 h light and 12 h dark’ cycles and fed with normal pellet dietand water ad libitum.

After the acclimatization period of 10 days, blood samples werecollected from marginal ear vein of all the rabbits. The blood wasallowed to clot and then centrifuged at 3000 rpm for 10 min and theserum was carefully drawn and collected into separate tubes. The serumwas analyzed for total cholesterol, triglycerides, high densitylipoproteins (HDL), low density lipoproteins (LDL) and very low densitylipoproteins (VLDL) levels using auto-analyzer.

After taking baseline lipid profile the animals were divided into fourgroups having six animals in each group. Following treatment was givento the animals for the three months:

TABLE 8 Treatment schedule. Groups Drugs administered Group I Vehiclefor 3 months. Group II Cholesterol (100 mg/kg) + Vehicle for 3 months.Group III Cholesterol (100 mg/kg) + Amla seed extract as per example 1(10 mg/kg) for 3 months. Group IV Cholesterol (100 mg/kg) + Atorvastatin(10 mg/kg) for 3 months.

Blood samples were collected from all the rabbits after 3 months oftreatment and serum was analyzed for lipid profile. After bloodcollection the animals were sacrificed after injecting pentobarbitoneand aorta was dissected out, washed with saline and preserved in 10%formalin for histopathology. The sections were cut using microtome andstained with heamatoxylin & eosin dye and mounted on glass slides. Theslides were observed under microscope and aortic intima media thickness(IMT) was measured by histomorphometry.

TABLE 9 (A) Lipid profile (mg/dl) of rabbits treated with amla seedextract. TC TG HDL Groups Before After Before After Before After Normalcontrol 58.23 56.77 62.25 60.05 16.81 15.89 (vehicle only).Cholesterol + 54.88 219.81 59.26 188.25 14.58 11.5 vehicle.Cholesterol + 56.36 94.18 60.61 83.23 15.42 37.27 amla seed extract.Cholesterol + 53.69 118.9 58.28 78.67 14.15 20.37 atorvastatin.

TABLE 9 (B) Lipid profile (mg/dl) of rabbits treated with amla seedextract. LDL VLDL Groups Before After Before After Normal control(vehicle only). 28.97 28.37 12.45 12.01 Cholesterol + vehicle. 28.45170.66 11.85 37.65 Cholesterol + amla seed extract. 28.82 40.26 12.1216.65 Cholesterol + atorvastatin. 27.88 82.8 11.66 15.73

TABLE 10 Aortic intima media thickness (IMT) of rabbits treated withamla seed extract. Aortic intima media thickness Groups (μm) Normalcontrol (vehicle only). 49.52 Cholesterol + vehicle. 123.24Cholesterol + amla seed extract. 66.21 Cholesterol + atorvastatin.112.36

Results show that, before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Feeding cholesterol for three months increased the totalcholesterol and triglyceride levels to very high.

Administration of Cholesterol alone (Group II) significantly increasedthe total cholesterol level to 219.81 mg/dl. Whereas in Group III, oraladministration of cholesterol+ amla seed extract, total cholesterollevel was 94.18 mg/dl which was 2.3 times lower as compared tocholesterol alone group.

In group II cholesterol administration increased the triglyceride levelto 188.25 mg/dl. Whereas in Group III, oral administration ofcholesterol+ amla seed extract, triglyceride level was 83.23 mg/dl whichwas 2.3 times lower as compared to cholesterol alone group.

In group III, administration of cholesterol+ amla seed extract increasedthe HDL (good cholesterol) level significantly from a baseline value of15.42 to 37.27 after 3 months treatment. That was a 2.4 times increasein the HDL cholesterol after 3 month treatment.

After cholesterol administration the cholesterol alone group showed aratio of HDL to total cholesterol was 0.05. Whereas in Group III, oraladministration of cholesterol+ amla seed extract, the ratio of HDLcholesterol to total cholesterol was 0.39 which was 7.8 times higher ascompared to cholesterol alone group.

In group II administration of cholesterol alone increased the LDL levelto 170.66 mg/dl. Whereas in Group III, oral administration ofcholesterol+ amla seed extract, LDL level was 40.26 mg/dl which was 4.2times lower as compared to triton alone group.

In group II administration of cholesterol alone increased the VLDL levelto 37.65 mg/dl. Whereas in Group III, oral administration ofcholesterol+ amla seed extract, VLDL level was 16.65 mg/dl which was 2.3times lower as compared to cholesterol alone group.

Atorvastatin also decreased the total cholesterol and triglyceride levelsignificantly as compared to untreated control group but amla seedextract was better especially in increasing the HDL level.

Moreover, administration of amla seed extract showed a 1.9 timesreduction in the intima media thickness of aorta of rabbits as comparedto untreated control group.

Example 24 Hair Fall Prevention and Hair Growth Promoting Activity ofAmla Seed Extract in Humans

Method:

10 human subjects suffering with alopecia and severe hair fall (asdetected by a dermatologist) were randomly divided into two groupshaving 5 subjects in each group.

Group I—Amla seed extract group prepared as per example 1.

Group II—Placebo group.

All the subjects were prohibited to take any kind of medicines (oral ortopical) having hair growth promotion like minoxidil, finasteride etc.for one month prior to the study initiation. The subjects of group Iapplied 5 ml coconut oil containing 5% amla seed extract twice daily onthe affected area and also took 100 mg of amla seed extract capsulestwice daily. The subjects of placebo group were given coconut oil(without amla seed extract) to be applied twice daily on affected areaand were provided with placebo capsules to be taken twice daily. Thetreatment was continued for 3 months and observations were taken by adermatologist before and after the study period. Length and thickness ofrandomly plucked five hairs from each subject was also determined.

Results:

The hairs of subjects treated with amla seed extract were shiny,lustrous and denser as compared to placebo group. The hair fall wasalmost arrested in the subjects treated with amla seed extract. Incontrast, the subjects of placebo group observed hair fall at the samerate as it was before starting the treatment. The average length ofhairs of subjects treated with amla seed extract was about 25% more thanthe subjects of placebo group. Thickness of the hairs was also about 20%more in amla seed extract group as compared to subjects in placebogroup. Therefore, amla seed extract was helpful in decreasing the hairfall as well as in promoting hair growth. Amla seed extract also madethe hairs lustrous and shiny.

Example 25 Hypolipidemic Activity of Amla Seed Extract Compared withOther Amla Extracts Using Triton WR 1339 Induced Dyslipidemia Model

Forty eight male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intotwelve groups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only).

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal).

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by ethylacetate of methanol extract of amla seed prepared as per example 1 (2.5mg/kg, per oral).

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterpart of methanol extract of amla seed prepared as per example 1 (2.5mg/kg, per oral).

Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by ethylacetate extract of amla seed prepared as per example 2 (2.5 mg/kg, peroral).

Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by powderof dried amla seed prepared as per example 7 (2.5 mg/kg, per oral).

Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterextract of dried amla seed powder prepared as per example 8 (2.5 mg/kg,per oral).

Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed bymethanol extract of dried amla seed powder prepared as per example 9(2.5 mg/kg, per oral).

Group IX: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by powderof dried amla fruit prepared as per example 10 (2.5 mg/kg, per oral).

Group X: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterextract of dried Amla fruit powder prepared as per example 11 (2.5mg/kg, per oral).

Group XI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed bymethanol extract of dried amla fruit powder prepared as per example 12(2.5 mg/kg, per oral).

Group XII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byatorvastatin (2.5 mg/kg, per oral).

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 11(A) Lipid profile (mg/dl) of rats treated with amla seed extractand amla fruit extracts in same dosage level. TC TG HDL Groups BeforeAfter Before After Before After Normal control. 90.15 94.16 58.18 61.6530.12 31.01 Triton only. 98.92 288.18 60.15 581.42 35.59 42.62 Triton +EtOAc extract amla 101.12 106.41 62.42 90.47 34.14 48.15 seed preparedas per example 1 (2.5 mg/Kg). Triton + water part of amla seed 102.54266.25 54.57 556.35 32.24 37.54 prepared as per example 1 (2.5 mg/kg).Triton + EtOAc extract of amla 99.35 122.25 55.87 99.5 36.25 42.51 seedprepared as per example 2 (2.5 mg/kg). Triton + powder of dried amla97.54 275.56 58.65 541.35 31.25 36.65 seed prepared as per example 7(2.5 mg/kg). Triton + water extract of amla 89.2 257.6 46 548 31 34 seedpowder prepared as per example 8 (2.5 mg/kg). Triton + methanol extractof 96.55 251.64 55.69 552.36 35.68 39.41 amla seed powder prepared asper example 9 (2.5 mg/kg). Triton + powder of dried amla 95.64 253.1459.68 575.36 33.54 37.74 fruit prepared as per example 10 (2.5 mg/kg).Triton + water extract of amla 101.22 265.33 59.85 547.96 31.54 41.52fruit powder prepared as per example 11 (2.5 mg/kg). Triton + methanolextract of 99.64 254.39 60.77 577.63 34.88 40.57 amla seed powderprepared as per example 12 (2.5 mg/kg). Triton + Atorvastatin 104.44123.14 49.54 195.25 36.54 39.56 (2.5 mg/kg).

TABLE 11(B) Lipid profile (mg/dl) of rats treated with amla seed extractand amla fruit extracts in same dosage level. LDL VLDL Groups BeforeAfter Before After Normal control. 48.39 50.82 11.64 12.33 Triton only.51.3 129.28 12.03 116.28 Triton + EtOAc extract amla seed prepared as54.5 40.17 12.48 18.09 per example 1 (2.5 mg/Kg). Triton + water part ofamla seed prepared as 59.39 117.44 10.91 111.27 per example 1 (2.5mg/kg). Triton + EtOAc extract of amla seed prepared 51.93 59.84 11.1719.9 as per example 2 (2.5 mg/kg). Triton + powder of dried amla seedprepared 54.56 130.64 11.73 108.27 as per example 7 (2.5 mg/kg).Triton + water extract of amla seed powder 59.16 131.86 12.25 114.29prepared as per example 8 (2.5 mg/kg). Triton + methanol extract of amlaseed 49.73 101.76 11.14 110.47 powder prepared as per example 9 (2.5mg/kg). Triton + powder of dried amla fruit prepared 50.16 100.33 11.94115.07 as per example10 (2.5 mg/kg). Triton + water extract of amlafruit powder 57.71 114.22 11.97 109.59 prepared as per example 11 (2.5mg/kg). Triton + methanol extract of amla seed 52.61 98.29 12.15 115.53powder prepared as per example 12 (2.5 mg/kg). Triton + Atorvastatin(2.5 mg/kg). 57.99 44.53 9.91 39.05

In this study, amla seed extract (2.5 mg extract/kg body weight) made inexample 1 and 2 were compared with other amla extracts made as inexample 7 to 12 at the same dose level, i.e. 2.5 mg of extract/kg bodyweight.

Results show that, before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 288.18 mg/dl. Whereas in Group III, simultaneousoral administration of amla seed extract prepared as per example 1 intriton injected rats, cholesterol level was 106.41 mg/dl which was 2.7times lower as compared to triton alone group.

In group II triton injection increased the triglyceride level to 581.42mg/dl. Whereas in Group III, simultaneous oral administration of amlaseed extract prepared as per Example 1 in triton injected rats,triglyceride level was 90.47 mg/dl which was 6.4 times lower as comparedto triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.15. Whereas in Group III, simultaneous oraladministration of amla seed extract prepared as per example 1 in tritoninjected rats, the ratio of HDL cholesterol to total cholesterol was0.45 which was 3 times higher as compared to triton alone group.

In group II triton injection increased the LDL level to 129.28 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract (prepares as per example 1) in triton injected rats, LDL levelwas 40.17 mg/dl which was 3.2 times lower as compared to triton alonegroup.

In group II triton injection increased the VLDL level to 116.28 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract (prepared as per example 1) in triton injected rats, VLDL levelwas 18.09 mg/dl which was 6.4 times lower as compared to triton alonegroup.

The ethyl acetate extract of amla seeds (prepared in example 2) at 2.5mg/kg were also able to decrease the cholesterol and triglyceride levelto a certain extent (Group V).

Results show that, before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 288.18 mg/dl. Whereas in Group III, simultaneousoral administration of amla seed extract prepared as per example 2 intriton injected rats, cholesterol level was 122.25 mg/dl which was 2.3times lower as compared to triton alone group.

In group II triton injection increased the triglyceride level to 581.42mg/dl. Whereas in Group III, simultaneous oral administration of amlaseed extract prepared as per Example 2 in triton injected rats,triglyceride level was 99.5 mg/dl which was 5.8 times lower as comparedto triton alone group.

In triton alone group after injection the ratio of HDL to totalcholesterol was 0.15. Whereas in Group III, simultaneous oraladministration of amla seed extract prepared as per example 2 in tritoninjected rats, the ratio of HDL cholesterol to total cholesterol was0.35 which was 2.3 times higher as compared to triton alone group.

In group II triton injection increased the LDL level to 129.28 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract (prepares as per example 2) in triton injected rats, LDL levelwas 59.84 mg/dl which was 2.1 times lower as compared to triton alonegroup.

In group II triton injection increased the VLDL level to 116.28 mg/dl.Whereas in Group III, simultaneous oral administration of amla seedextract (prepared as per example 2) in triton injected rats, VLDL levelwas 19.9 mg/dl which was 5.8 times lower as compared to triton alonegroup.

The water part of methanol extract (prepared in example 1), amla seedpowder and other water and methanol extracts (prepared in example 7 to12) were not active in reducing cholesterol or triglyceride levels.Though the standard drug atorvastatin was effective in lowering thecholesterol as well as triglyceride levels (Group XII) but lesseffective when compared with the ethyl acetate part of amla seedmethanol extract (as per example 1) or ethyl acetate extract of amlaseeds (as per example 2).

Example 26 Hypolipidemic Activity of Amla Seed Extract (Product 3)Compared with Amla Seed Powder Using Triton WR 1339 Induced DyslipidemiaModel

Twenty eight male Albino rats (Sprague Dawley strain) weighingapproximately 250-300 gm were selected for the study. The animals weremaintained at temperature of 24±2° C., 65% relative humidity and 12 hrlight/dark cycle. The rats were acclimatized for two weeks and duringthis period they had access to standard pellet diet and water adlibitum. After two weeks of acclimatization, all the rats were fastedovernight before injecting Triton WR 1339 (Tyloxapol) and administrationof test extracts/standard. The animals were divided into seven groups.Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal)

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byadministering product of amla seed extract (product 3) on the same dayof triton administration (0.5 mg/kg, per oral)

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byproduct 2 (50% methanol fraction+ 80% methanol fraction+ water fraction)of amla seed extract on the same day that Triton was administered (0.5mg/kg, per oral)

Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by product1 (Hexane fraction) of amla seed extract on the same day that Triton wasadministered (0.5 mg/kg, per oral)

Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by powderof dried amla seed prepared as per Example 7 (0.5 mg/kg, per oral). Thepowder of dried amla seed was administered on the same day as triton wasadministered.

Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byatorvastatin administration (10 mg/kg, per oral), on the same day.

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hours of drug treatment, 2 ml of bloodsample was collected from the retro orbital plexus. The blood wasallowed to clot and then centrifuged at 3000 rpm for 10 min and theserum was carefully drawn and collected into separate tubes. The serumwas analyzed for total cholesterol, triglycerides, high densitylipoproteins (HDL), low density lipoproteins (LDL) and very low densitylipoproteins (VLDL) levels using auto-analyzer.

TABLE 12(A) Lipid profile (mg/dl) of rats treated with amla seedextracts (product 3) and different fractions (product 1 and product 2).TC TG HDL Groups Before After Before After Before After Group I 91.0595.11 57.16 60.47 29.31 30.11 Normal control. Group II 109.55 386.4 49.5583.33 32.5 43.2 Triton only. Group III 90.2 95.6 44.66 62.4 28.78 49.36Triton + amla seed product 3 (0.5 mg/Kg). Group IV 96.5 132.36 46.8103.5 27.4 41.03 Triton + amla seed product 2 (0.5 mg/Kg). Group V 95.7184.5 50.12 119.4 30.9 47.97 Triton + amla seed product 1 (0.5 mg/Kg).Group VI 96.85 345.6 59.65 542.54 32.35 34.45 Triton + powder of driedamla seed prepared as per Example 7 (0.5 mg/kg). Group VII 103.47 121.2148.54 192.25 35.54 38.46 Triton + atoryastatin (10 mg/kg).

TABLE 12(B) Lipid profile (mg/dl) of rats treated with Amla seedextracts (product 3) and different fractions (Product 1 and product 2).LDL VLDL Groups Before After Before After Group I 50.31 52.91 50.3152.91 Normal control Group II 67.15 226.53 67.15 226.53 Triton onlyGroup III 60.81 33.76 60.81 33.76 Triton + amla seed product 3 (0.5mg/Kg) Group IV 62.3 70.63 62.3 70.63 Triton + amla seed product 2 (0.5mg/Kg) Group V 65.4 112.65 65.4 112.65 Triton + amla seed product 1 (0.5mg/Kg) Group VI 52.57 202.64 52.57 202.64 Triton + powder of dried amlaseed prepared as per example 7 (0.5 mg/kg) Group VII 58.22 44.30 58.2244.30 Triton + atorvastatin (10 mg/kg)

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 386.4 mg/dl. Whereas in Group III, following oraladministration of product 3 of amla seed extract to triton injectedrats, the cholesterol level was 95.6 mg/dl. Therefore, administeringproduct 3 (Group III) resulted in 4 times lower level of cholesterol ascompared to triton alone treatment (Group II).

In Group II triton injection increased the triglyceride level to 583.33mg/dl. Whereas in Group III, oral administration of product 3 to tritoninjected rats, the triglyceride level was 62.4 mg/dl, which was 9.3times lower as compared to triton alone group (Group 2).

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group III, the ratio of HDL cholesterolto total cholesterol was 0.51, which was 4.6 times higher as compared totriton alone group.

In group II triton injection increased the LDL level to 226.53 mg/dl.Whereas in Group III, LDL level was 33.76 mg/dl, which was 6.7 timeslower as compared to triton alone group.

In group II triton injection increased the VLDL level to 116.67 mg/dl.Whereas in Group III, the VLDL level was 12.48 mg/dl, which was 9.3times lower as compared to triton alone group.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 386.4 mg/dl. Whereas in Group IV (Triton followedby Product 2 administration), cholesterol level was 132.36 mg/dl, whichwas 2.9 times lower as compared to triton alone group.

In Group II Triton injection increased the triglyceride level to 583.33mg/dl. Whereas in Group IV, oral administration of product 2 to tritoninjected rats, triglyceride level was 103.5 mg/dl, which was 5.6 timeslower as compared to triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group IV, oral administration of amlaseed extract (product 2) to triton injected rats, the ratio of HDLcholesterol to total cholesterol was 0.30 which was 2.7 times higher ascompared to triton alone group.

In Group II, Triton injection increased the LDL level to 226.53 mg/dl.Whereas in Group IV, oral administration of product 2 to triton injectedrats, LDL level was 70.63 mg/dl, which was 3.2 times lower as comparedto triton alone group.

In Group II, triton injection increased the VLDL level to 116.67 mg/dl.Whereas in Group IV, oral administration of product 2 to triton injectedrats, VLDL level was 20.7 mg/dl, which was 5.6 times lower as comparedto triton alone group (Group II).

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 386.4 mg/dl. Whereas in Group V, simultaneous oraladministration of product 1 prepared as per example 13 in tritoninjected rats, cholesterol level was 184.5 mg/dl which was 2.09 timeslower as compared to triton alone group.

In group II triton injection increased the triglyceride level to 583.33mg/dl. Whereas in Group V, simultaneous oral administration of product 1in triton injected rats, triglyceride level was 119.4 mg/dl which was4.9 times lower as compared to triton alone group.

In triton alone group after injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group V, simultaneous oraladministration of product 1 prepared as per example 13 in tritoninjected rats, the ratio of HDL cholesterol to total cholesterol was0.26 which was 2.4 times higher as compared to triton alone group.

In group II triton injection increased the LDL level to 226.53 mg/dl.Whereas in Group V, simultaneous oral administration of product 1prepared as per example 13 in triton injected rats, LDL level was 112.65mg/dl which was 2 times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 116.67 mg/dl.Whereas in Group V, simultaneous oral administration of product 1prepared as per example 13 in triton injected rats, VLDL level was 23.88mg/dl which was 4.9 times lower as compared to triton alone group.

Example 27 Method of Preparation of Extract of Seed of Emblicaofficinalis Enriched with Triterpenoids

500 Kg of fresh fruits of Emblica officinalis (Amla) were collected.Fruits were deseeded by deseeding machine and 75 Kg of fresh seeds werecrushed through roller mill. 95% Methanol in an amount 2 times thequantity of crushed seeds was added to the crushed seeds to form amixture for methanol extraction. The extraction was performed using anextractor with reflux condenser. The bottom of the extractor was fittedwith a polypropylene (100 microns) filter cloth. The mixture wasrefluxed for one hour at 65° C. to obtain a residue and a firstsupernatant. The residue and the first supernatant were separated bydraining out the supernatant from the extractor bottom through thepolypropylene filter cloth using a centrifugal pump. After the firstextraction, the remaining residue was further extracted with two timesthe quantity of methanol at 65° C. to again obtain a residue and asecond supernatant. This residue was further extracted with two timesthe quantity of methanol at 65° C. to obtain another residue and a thirdsupernatant. The first, second and third supernatants were pooled andthe resulting mixture was concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form a concentratedmethanol extract. The concentrated methanol extract was dried undervacuum at above 500 mm of mercury to obtain 5 kg of powder. This powderis referred to as methanol extract powder of seed of Emblicaofficinalis.

The methanol extract powder of seed of Emblica officinalis was maceratedwith water and transferred into a liquid-liquid extractor and extractedwith ethyl acetate. Ethyl acetate phase and aqueous phase wereseparated. After extraction ethyl acetate phase was collected. Ethylacetate phase was concentrated in an Agitated thin film evaporator toform concentrated ethyl acetate extract. Ethyl acetate concentrate wasfed into vacuum stripper and dried under vacuum at above 500 mm ofmercury to obtain 2.5 kg of powder. This powder is referred to as ethylacetate extract powder from methanol extract powder of seed of EmblicaOfficinalis.

50% Methanol (10 L) was added to 2.5 kg of ethyl acetate extract powderfrom methanol extract of seed of Emblica officinalis to form a mixturefor methanol extraction. The extraction was performed using an extractorwith reflux condenser. After refluxing the oil part formed was separatedfrom the methanol-water part. Oil part was discarded and methanol-waterpart was concentrated to form concentrated methanol part with a yield of1.8 Kg.

Concentrated methanol-water part was loaded on a HP20 column. Column waseluted with ethyl acetate and 100% methanol. Each fraction was collectedand concentrated. Concentrated methanol fraction was dried under vacuumto form powder of seed of Emblica officinalis with a yield of 0.8 kg.This powder was referred to as Sample 1. Sample 1 was found to haveabout 63.5% triterpenoids and about 27% hydroxycinnamic acids.

A blend was prepared with Sample 1 as the first extract of seed ofEmblica officinalis and a second extract of amla seed (also referred toas product 1) which was attained by the method of Example 13. A methodof preparing the first extract of seeds of Emblica officinalis isprovided in FIG. 14A. Method for preparing second extract of amla seedand blend of the first and second extract of seeds of Emblicaofficinalis is provided in FIG. 14B.

0.8 Kg of powder of seed of Emblica officinalis (sample 1) was combinedwith 0.8 Kg of product 1 prepared in example 13 in a 1:1 ratio to forman amla seed blend with a yield of 1.6 Kg. The blend is also referred toas Sample 2.

Sample 2 had about 32% triterpenoids, about 13.5% hydroxycinnamic acids,about 38.6% unsaturated fatty acids and about 0.3% saturated fattyacids.

Example 28 Hypolipidemic Activity of Enriched Amla Seed Extract Comparedwith Other Amla Extract Using Triton WR 1339 Induced Dyslipidemia Model

Fifty two male Albino rats (Sprague Dawley strain) weighingapproximately 250-300 gm were selected for the study. The animals weremaintained at temperature of 24±2° C., 65% relative humidity and 12 hrlight/dark cycle. The rats were acclimatized for two weeks and duringthis period they had access to standard pellet diet and water adlibitum. After two weeks of acclimatization, all the rats were fastedovernight before injecting Triton WR 1339 (Tyloxapol) and administrationof test extracts/standard. The animals were divided into thirteengroups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only).

Group II: Triton WR 1339 (300 mg/kg, intraperitoneal).

Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byadministering sample 1 of Amla seed extract prepared as per example 27on the same day of triton administration (0.5 mg/kg, per oral).

Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepared as per example 1 (0.5 mg/kg, per oral).

Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by sample2 which is a 1:1 combination of amla seed extract (sample 1) prepared asper example 27 and product 1 (Hexane fraction) of amla seed extractprepared as per example 13 on the same day that Triton was administered(0.5 mg/kg, per oral).Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byadministering product of amla seed extract (product 3) prepared as perexample 13 on the same day of triton administration (0.5 mg/kg, peroral).Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byproduct 2 (50% methanol fraction+ 80% methanol fraction+ water fraction)of amla seed extract prepared as per example 13 on the same day thattriton was administered (0.5 mg/kg, per oral).Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byproduct 1 (hexane fraction) of amla seed extract prepared as per example13 on the same day that triton was administered (0.5 mg/kg, per oral).Group IX: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterextract of dried amla seed powder prepared as per example 8 (0.5 mg/kg,per oral).Group X: Triton WR 1339 (300 mg/kg, intraperitoneal) followed bymethanol extract of dried amla seed powder prepared as per example 9(0.5 mg/kg, per oral).Group XI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterextract of dried amla fruit powder prepared as per example 11 (0.5mg/kg, per oral).Group XII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed bymethanol extract of dried amla fruit powder prepared as per example 12(0.5 mg/kg, per oral).Group XIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byatorvastatin administration (10 mg/kg, per oral), on the same day.

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hours of drug treatment, 2 ml of bloodsample was collected from the retro orbital plexus. The blood wasallowed to clot and then centrifuged at 3000 rpm for 10 min and theserum was carefully drawn and collected into separate tubes. The serumwas analyzed for total cholesterol, triglycerides, high densitylipoproteins (HDL), low density lipoproteins (LDL) and very low densitylipoproteins (VLDL) levels using auto-analyzer.

TABLE 13(A) Lipid profile (mg/dl) of rats treated with different Amlaseed extracts. TC TG HDL Groups Before After Before After Before AfterGroup I 90.6 94.4 55 61 31.9 30.5 Normal control Group II 100.32 397.447.6 545 30.5 41.8 Triton only Group III 82.04 97.98 49.2 104.9 30.243.2 Triton + Amla seed sample 1 (0.5 mg/Kg) Group IV 94.76 116.06 50.3125.3 29.3 39 Triton + Amla seed extract prepared as per example 1 (0.5mg/Kg) Group V 91.44 91.1 48.2 64.5 30.1 48.5 Triton + Amla seed (sample2) (0.5 mg/Kg) Group VI 90.46 97 46.8 70 29.6 46 Triton + Amla seed(produc t3) (0.5 mg/kg) Group VII 95.64 134.55 47.2 109.8 27.2 37.09Triton + Amla seed (product 2) (0.5 mg/kg) Group VIII 93.58 251.8 47.9124 28 32 Triton + Amla seed (product 1) (0.5 mg/kg) Group IX 89.2 257.646 548 31 34 Triton + water extract of dried Amla seed powder (0.5mg/kg). Group X 92.6 245.2 43.1 521 33 35 Triton + methanol extract ofdried Amla seed powder (0.5 mg/kg). Group XI 87.8 240.2 43 546 27.2 31Triton + water extract of dried Amla fruit powder (0.5 mg/kg). Group XII90.4 248.8 42 559 29 33 Triton + methanol extract of dried Amla fruitpowder (0.5 mg/kg). Group XIII 104.21 124.54 50.54 200.25 32.41 35.1Triton + Atorvastatin (10 mg/kg)

TABLE 13(B) Lipid profile (mg/dl) of rats treated with different Amlaseed extracts. LDL VLDL Groups Before After Before After Group I 47.751.7 11 12.2 Normal control Group II 60.3 246.6 9.52 109 Triton onlyGroup III 42 33.8 9.84 20.98 Triton + Amla seed sample 1 (0.5 mg/Kg)Group IV 55.4 52 10.06 25.06 Triton + Amla seed extract prepared as perexample 1 (0.5 mg/Kg) Group V 51.7 29.7 9.64 12.9 Triton + Amla seedsample 2 (0.5 mg/Kg) Group VI 51.5 37 9.36 14 Triton + Amla seed(product 3) (0.5 mg/kg) Group VII 59 75.5 9.44 21.96 Triton + Amla seed(product 2) (0.5 mg/kg) Group VIII 56 195 9.58 24.8 Triton + Amla seed(product 1) (0.5 mg/kg) Group IX 49 114 9.2 109.6 Triton + water extractof dried Amla seed powder (0.5 mg/kg). Group X 51 106 8.6 104.2 Triton +methanol extract of dried Amla seed powder (0.5 mg/kg). Group XI 52 1008.6 109.2 Triton + water extract of dried Amla fruit powder (0.5 mg/kg).Group XII 53 104 8.4 111.8 Triton + methanol extract of dried Amla fruitpowder (0.5 mg/kg). Group XIII 61.7 49.39 10 40.06 Triton + Atorvastatin(10 mg/kg)

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 397.4 mg/dl, whereas in Group V, following oraladministration of blend of Amla seed extract (sample 1) and product 1 totriton injected rats, the cholesterol level remained about 91.1 mg/dleven after 24 hours of testing. Therefore, administering Group Vresulted in 4.4 times lower level of cholesterol as compared to tritonalone treatment (Group II).

In Group II triton injection increased the triglyceride level to 545mg/dl. Whereas in Group V, oral administration of blend of amla seedextract (sample 1) and product 1 to triton injected rats, thetriglyceride level was 64.5 mg/dl, which is 8.5 times lower as comparedto triton alone group (Group 2).

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group V, the ratio of HDL cholesterolto total cholesterol was 0.53, which is 4.8 times higher as compared totriton alone group.

In group II, triton injection increased the LDL level to 246.6 mg/dl.Whereas in Group V, LDL level was 29.7 mg/dl, which is 8.3 times loweras compared to triton alone group.

In group II, triton injection increased the VLDL level to 109 mg/dl.Whereas in Group V, the VLDL level was 12.9 mg/dl, which is 8.5 timeslower as compared to triton alone group.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 397.4 mg/dl. Whereas in Group VI (Triton followedby product 3 administration), cholesterol level was 97 mg/dl, which is 4times lower as compared to triton alone group.

In Group II Triton injection increased the triglyceride level to 545mg/dl. Whereas in Group VI, oral administration of product 3 to tritoninjected rats, triglyceride level was 70 mg/dl, which is 7.7 times loweras compared to triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group VI, oral administration of amlaseed extract (product 3) to triton injected rats, the ratio of HDLcholesterol to total cholesterol was 0.47 which is 4.2 times higher ascompared to triton alone group.

In Group II, Triton injection increased the LDL level to 246.6 mg/dl.Whereas in Group VI, oral administration of Product 3 to triton injectedrats, LDL level was 37 mg/dl, which is 6.6 times lower as compared totriton alone group.

In Group II, triton injection increased the VLDL level to 109 mg/dl.Whereas in Group VI, oral administration of product 3 to triton injectedrats, VLDL level was 14 mg/dl, which is 7.8 times lower as compared totriton alone group (Group II).

In group IX, X, XI and XII, oral administration of water extract of Amlafruit and seed, and methanol extract of amla fruit and seed showedslight activity in reducing total cholesterol, triglyceride, HDL, LDLand VLDL. In comparison to activity in Group V, the activity in group IXto XII was negligible.

Group V, fed with a blend of amla seed extract (sample 1) and product 1has shown a significantly higher efficacy in comparison to oil part ofthe seed, that is Group VIII, as well as methanol extract of seed as inGroup X.

Example 29 Effect of Amla Seed Extract in Patients with Dyslipidaemia

A total of 50 subjects were divided into two groups of 25 subjects each.Both male and female patients having Triglycerides (TG) greater than 200mg/dL, LDL cholesterol greater than 130 mg/dL, total cholesterol greaterthan 200 mg/dL and HDL-C below 40 mg/dL for men and 50 mg/dL for womenwere selected for the study. Patients were not taking any medication(including herbal product) for management of dyslipidaemia. The subjectswere given one 500 mg capsule of Amla seed extract prepared as perexample 1 or placebo daily after food for 12 weeks.

Efficacy of the study medication was assessed by serum lipid parametersincluding triglycerides, total Cholesterol, LDL and VLDL. Otherparameters measured were fasting plasma glucose, glycosylatedhaemoglobin, Apo lipoproteins A1, Apo lipoproteins B, homocysteine,Coenzyme Q10, high sensitivity C-reactive protein (hs-CRP), HMG CoAreductase inhibitory activity and TSH.

TABLE 1 Lipid profile (mg/dl) of subjects treated with Amla seed extractand Placebo. Amla seed extract Placebo After After Parameters Baseline12 weeks Baseline 12 weeks Total cholesterol (mg/dl) 230 177 224 222Triglycerides (mg/dl) 261 150 244 245 High Density 43 55 43 42Lipoproteins (mg/dl) Low Density 136 92 132 131 Lipoproteins (mg/dl)Very Low Density 52 30 49 49 Lipoproteins (mg/dl) Atherogenic index of0.44 0.09 0.41 0.42 Plasma

Twelve weeks treatment with amla seed extract decreased the totalcholesterol 23% when compared with baseline value whereas in placebogroup the reduction was near to only 1%. In case of triglycerides thereduction was 43% in amla seed extract group whereas in placebo group,reduction in triglycerides was 0.4% only. There was significant increaseof 28% in HDL level of amla seed extract group whereas HDL level ofplacebo group was decreased by 2%. Decrease in LDL was very significantand it was 32% in amla seed extract group whereas in placebo thedecrease in LDL was 0.8% only. VLDL was decreased about 42% in amla seedextract group whereas in placebo group there was no difference in VLDLlevel compared to baseline.

As a marker of plasma atherogenecity Atherogenic index of the plasma(AIP) is used, AIP is the logarithmically transformed ratio of molarconcentrations of triglycerides (TGs) to HDL-cholesterol (log (TG/HDL[mmol]). AIP increases in people at higher risk for coronary heartdisease and is inversely correlated with LDL particle size.

AIP values of −0.3 to 0.1 are associated with low, 0.1 to 0.24 withmedium and above 0.24 with high cardiovascular risk. In present studyreduction in AIP is observed from 0.44 to 0.09 in amla seed extractgroup whereas in placebo group very minor increase in AIP is observed.That is amla seed extract decreased the atherogenic index of plasma by80% in 12 weeks treatment whereas in placebo group the change in AIP wasonly 2%.

TABLE 2 Apo lipoprotein A1, Apo lipoprotein B, hs-CRP, Homocysteine,Glycosylated Haemoglobin, TSH, HMGCoA reductase inhibitory activity andCoQ10 of subjects treated with Amla seed extract and Placebo. GroupsAmla Seed extract Placebo After After Parameters Baseline 12 weeksBaseline 12 weeks Apo lipoprotein A1g/L 1.05 1.32 1.01 1.02 Apolipoprotein B g/L 1.52 1.11 1.49 1.48 Ratio of Apo lipoprotein 1.45 0.841.48 1.45 B to Apo lipoprotein A1 hs-CRP mg/L 3.83 2.56 3.95 4.0Homocysteine μmol/L 23.58 15.61 21.1 20.5 Glycosylated 6.70 5.59 6.356.35 Haemoglobin % TSH μIU/ml 2.43 3.19 2.53 2.45 HMGCoA reductase 62.454.8 63.4 63.4 inhibitory activity ng/ml CoQ10 ng/ml 47.3 46.9 48.7 49.6

Apo A-1 is a protein that has a specific role in the metabolism oflipids and is the main protein component in HDL, the “good cholesterol”.Deficiencies in Apo A-1 correlate with an increased risk of developingCardio vascular disease (CVD). The reference range of Apo-A1 varies bysex, as follows: Men: Greater than 120 mg/dL (1.2 g/L) and Women:Greater than 140 mg/dL (1.4 g/L). In this study, the Apo A-1 has beenincreased from 1.05 g/L to 1.32 g/L in amla seed extract group whereasin placebo group there was no change. That is an increase of 26% in ApoA-1 after 12 weeks of treatment with amla seed extract. This indicatesthe benefits of amla seed extract in reducing the risk of CVD.

Apolipoprotein B (Apo B) is an important component of many lipoproteinsthat are involved in atherosclerosis and cardiovascular disease.Usually, 85-90 percent of Apo B represents LDL particles. The referencerange of Apo B levels in adults is less than 130 mg/dL (1.3 g/L). Inthis study, the Apo B has been reduced from 1.52 g/L to 1.11 g/L in amlaseed extract group whereas in placebo group there was no change. Therewas a decrease of 27% in Apo B level in amla seed extract group whereasdecrease in placebo group was about 1%. This indicates the benefits ofamla seed extract in reducing the risk of CVD.

Ratio of the apoB/apoA1 is more effective at predicting heart attackrisk, than either the apoB or apoA1 measure alone. The normal values are0.5 to 1.0. In this study, the ratio of Apo B to Apo A-1 has beendecreased from 1.45 g/L to 0.84 g/L in amla seed extract group whereasin placebo group there was no change. That is ratio of Apo B to Apo A-1was decreased by 42% in amla seed extract group whereas the decrease wasonly 2% in placebo group. This indicates the benefits of amla seedextract in reducing the risk of CVD.

From the results it was observed that there was a decrease of 33% hs-CRPvalue of amla seed extract group whereas in placebo group hs-CRP wascomparable with baseline value.

Homocysteine is an amino acid and breakdown product of proteinmetabolism that, when present in high concentrations, has been linked toan increased risk of heart attacks and strokes. Homocysteine levels aretypically higher in men than women, and increase with age. Common levelsare in range of 10-15 μmol/L in plasma. In our study, the homocysteinelevel has been reduced from 23.58 to 15.61 μmol/L in amla seed extractgroup whereas in placebo group, there was no change as compared tobaseline value. That is twelve weeks treatment with amla seed extractdecreased the homocysteine level 33% when compared with baseline valuewhereas in placebo group the reduction was only about 2%. This indicatesthe benefits of amla seed extract in managing cardiovascular health andpreventing the risk of stroke, heart attack and other related risks.

HbA1c is a measure of the beta-N-1-deoxy fructosyl component ofhemoglobin. Normal levels of glucose produce a normal amount of glycatedhemoglobin. Excessive formation of early glycation products mayadversely affect several functions of blood vessels, lipid metabolismand prone to develop diabetic complications. HbA1c level below 5.7percent is considered normal whereas between 5.7 and 6.4 percent signalspre-diabetes. In our study, the level of HbA1c has been reduced from6.7% to 5.59% in amla seed extract group whereas in placebo group, therewas no change in HbA1c level. In case of glycosylated Hb the reductionwas 16% in amla seed extract group whereas there was no change inplacebo group when compared with the baseline value. This indicates thebenefits of amla seed extract in managing the blood sugar level andpreventing diabetes.

Thyroid function regulates a wide array of metabolic parameters. Thyroidfunction significantly affects lipoprotein metabolism as well as somecardiovascular disease (CVD) risk factors, thus influencing overall CVDrisk. Thyroid hormones induce the 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase, which is the first step in cholesterolbiosynthesis. Normal TSH level ranges from 0.4 to 4 μIU/ml. In ourstudy, the amla seed extract administration increased the TSH level from2.43 to 3.19, thus increasing the lipoprotein lipase which reflects indecreasing the TGs. In placebo group there was slight decrease in TSHlevel. Increase in the TSH level was 31% in amla seed extract groupwhereas TSH level of placebo group was decreased by 3%.

Coenzyme Q10 is the coenzyme for mitochondrial enzyme complexes involvedin oxidative phosphorylation in the production of ATP. A deficiency ofCoQ10 in the blood and the heart muscle has been documented incongestive heart failure. Primary and secondary deficiencies of CoQ10result in a number of neurologic and myopathic syndromes. HMG-CoAreductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase) is therate-controlling enzyme of the mevalonate pathway, the metabolic pathwaythat produces cholesterol and other isoprenoids. Normally in mammaliancells this enzyme is suppressed by cholesterol derived from theinternalization and degradation of low density lipoprotein (LDL) via theLDL receptor as well as oxidized species of cholesterol. In this study,highly significant reduction in HMGCoA reductase enzyme by Amla seedextract explains its mechanism of decreasing blood cholesterol andmanagement of cardio vascular health. At the same time, no change inCoQ10 level puts an advantage of Amla seed extract over statin therapyand makes it better choice than statins for treatment of dyslipidemia.

Decrease in HMGCoA reductase activity was 12% in amla seed extract groupand at the same time there was no change in CoQ10 level. There was nochange in HMGCoA reductase activity and CoQ10 level in placebo groupafter 12 weeks of treatment.

TABLE 3 Fasting Plasma Glucose (FPG) mg/dl of subjects treated with Amlaseed extract and Placebo. Groups Amla seed extract Placebo After AfterParameters Baseline 12 weeks Baseline 12 weeks Fasting Plasma Glucose90.6 84.4 91.9 100.4 mg/dl

Fasting plasma glucose (FPG) was decreased by 7% after 12 weeks oftreatment with amla seed extract whereas there was an increase of 9% inFPG of placebo group compared to baseline value.

Example 30 HMG-CoA Reductase Inhibition Assay of Amla Seed Extract

Amla seed extract prepared as per example 1 and water extract of driedAmla seed prepared as per example 8 was weighed separately and eachextract was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock of400 mg/ml stock solution of each. From this stock, different dose levelswere prepared in DMSO for further experimental use.

1× HMG-CoA Assay Inhibitor/test Reductase Sample buffer sample NADPHHMGCoA (HMGR) Blank 920 μl — 20 μl 60 μl — Activity 915 μl — 20 μl 60 μl5 μl Inhibition 910 μl 5 μl 20 μl 60 μl 5 μl

The optical density (OD) values were recorded using spectrophotometer atregular time intervals. Specific activity was calculated based on theobtained OD values using the following formula,

${{Units}\text{/}{mg}\mspace{14mu} P} = \frac{\left( {{{DA}\;{340/\min}\mspace{14mu}{sample}} - {{DA}\;{340/\min}\mspace{14mu}{blank}}} \right) \times {TV}}{12.44 \times V \times 0.6 \times {LP}}$Where:12.44=εmM—the extinction coefficient for NADPH at 340 nm is 6.22 mM-1cm−1. 12.44 represents the 2 NADPH consumed in the reaction.TV=Total volume of the reaction in ml (1 ml for cuvettes and 0.2 ml forplates)V=volume of enzyme used in the assay (ml)0.6=Enzyme concentration in mg-protein (mgP)/ml (0.50-0.70 mgP/ml)LP=Light path in cm (1 cm for cuvettes).

Based on the specific activity values from all the test doses, EC50(Effective Concentration 50) were determined by plotting a curve betweenSpecific activity Vs. Test concentration. EC50 values (μg/ml) and IC50(μM) were determined by interpolation method by using Graph pad prism,version 5.01.

TABLE 1 HMGR inhibitory activity of Amla seed extract. Test % Inhibitionover control Test % Inhibition over concentration Amla Seed Waterextract of concentration control (μg/ml) extract dried amla seed (μM)LOVASTATIN 200 98.51 66.57 100 98.51 100 91.49 49.37 80 86.56 50 47.1335.92 60 76.55 25 41.75 8.29 40 55.64 20 41.45 10 37.72 5 37.57 2.536.97 IC50 31.07 142.6 29.7

The IC50 value for amla seed extract was about 31 μM which was almostequivalent to the IC50 value (29.7 μM) of standard lovastatin. Thisindicates that amla seed extract has almost similar percentageinhibition of HMG CoA reductase as lovastatin. Water extract of driedamla seed was less effective and IC50 value was noted as 142.6 μM.

Example 31 Triterpenoid.Hydroxycinnamic Acid and Fatty Acid Ratios inAmla Seed Extracts

TABLE 1 Ratios of components in Amla seed extracts percentage FattyTriterpenoids Hydroxycinnamic acids Ratio (T) acid (H) (F) T:H:F Amlaseed powder Percentage 0.06%    0.3%   2% 1:5:33 EA extract of methanolPercentage 9.5%   4.3% 41.8% 2:1:10 extract of Amla seed [Example 1]product 1 (Example 13) Percentage 77.5% product 2 (Example 13)Percentage 50% 29.3% product 3 = Product 1 Percentage 20% 11.7% 46.5%2:1:4 blended with Product 2 in 3:2 ratio. [Example 13] Sample 1Percentage 63.5%     27% [Example 27] (Sample 2) Percentage 32% 13.5%38.6% 2:1:3 Amla seed extract enriched with triterpenoids i.e. Sample 1blended with Product 1 in 1:1 ratio [Example 27]

In amla seed powder, triterpenoids was about 0.06%, hydroxycinnamic acidwas about 0.3% and fatty acid was about 2%. Based on this, in amla seedpowder, the ratio of triterpenoids to hydroxycinnamic acid to fatty acidwas about 1:5:33. Fatty acid content was higher in the amla seed powdercompared to triterpenoids and hydroxycinnamic acid.

In the present product 3 (from Example 13), which was prepared byblending two extracts (product 1 from Example 13 and product 2 fromExample 13 in a ratio of 3:2), triterpenoids and hydroxycinnamic acidwere significantly enriched compared to fatty acids. The ratio oftriterpenoids to hydroxycinnamic acid to fatty acids in Product 3 wasabout 2:1:4, which is clearly different from the ratio found for amlaseed powder.

In amla seed blend composition (sample 2 from Example 27), the ratio oftriterpenoids to hydroxycinnamic acid to fatty acid was 2:1:3, which isvery different from the ratio obtained in amla seed powder.

Other modifications and variations to the invention will be apparent tothose skilled in the art from the foregoing disclosure and teachings.Thus, while only certain embodiments of the invention have beenspecifically described herein, it will be apparent that numerousmodifications may be made thereto without departing from the spirit andscope of the invention.

I claim:
 1. A method of lowering HMGCoA reductase in a subject in needthereof, the method comprising administering a blend of extracts ofseeds of Emblica officinalis, the blend comprising a first extract ofseeds of Emblica officinalis and a second extract of seeds of Emblicaofficinalis, the first extract comprising: a) about 20% to about 70%triterpenoids; and, b) about 10% to about 40% hydroxycinnamic acids,and, the second extract of seeds of Emblica officinalis comprising alphalinolenic acid, linoleic acid and oleic acid, wherein a weight ratio ofthe first extract of Emblica Officinalis to the second extract ofEmblica officinalis in the blend ranges from about 1:60 to about 99:1.2. A method of lowering HMGCoA reductase in a subject in need thereof,the method comprising administering an extract of seeds of Emblicaofficinalis, the extract comprising: about 0.5% to about 20%triterpenoids, about 0.5% to about 5% of hydroxycinnamic acids, and,about 25% to about 50% fatty acids.